AU2019347666B2

AU2019347666B2 - Cellular reprogramming to reverse aging and promote organ and tissue regeneration

Info

Publication number: AU2019347666B2
Application number: AU2019347666A
Authority: AU
Inventors: Yuancheng Lu; David A. Sinclair
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2018-09-28
Filing date: 2019-09-27
Publication date: 2025-10-02
Anticipated expiration: 2039-09-27
Also published as: US12414982B2; IL281470B1; JP2025032120A; US12409207B2; EP3856226A1; IL281470A; JP2022511373A; US20240316148A1; BR112021004670A2; WO2020069373A1; MX2021003663A; CN113453702A; AU2019347666A1; US20240261370A1; US20230338468A1; US20260014229A1; CA3113472A1; JP7593639B2; KR20210068485A; AU2025283586A1

Description

WO 2020/069373 A1 Published: Published: with with international international search search report report (Art. (Art. 21 (3)) 21(3))

- - with with sequence sequence listing listing part part of of description description (Rule (Rule 5.2(a)) 5.2(a))

- CELLULAR REPROGRAMMING TO REVERSE AGING AND PROMOTE ORGAN AND TISSUE REGENERATION RELATED APPLICATIONS

[0001] This application This application claims claims priority priority underunder 35 U.S.C. 35 U.S.C. § of § 119(e) 119(e) of U.S. provisional U.S. provisional

application number 62/738,922, filed September 28, 2018, U.S. provisional application

number 62/792,283, filed January 14, 2019, U.S. provisional application number 62/865,877,

filed June 24, 2019, and U.S. provisional application number 62/880,488, filed July 30, 2019,

each of which is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers R01

AG019719 and R01 DK100263 awarded by the National Institutes of Health (NIH). The

government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] In many animals, including vertebrates, vital organs have a limited intrinsic

capacity for regeneration and repair. Acute injury and chronic disorders can damage vital

organs and tissues, including the heart, pancreas, brain, kidney, muscles, skin and neuronal

tissue, among others. Mature somatic cells, however, often cannot survive these insults, and

even if they do, they are unable to self-renew and transdifferentiate to replace damaged cells.

Furthermore, cells that are capable of self-renewal can be limited in quantity, have limited

capacity and are susceptible to damage, especially with age. In contrast to somatic cells from

adults, cells from individuals that are chronologically closer to fertilization, such as those

from embryos and infants, display cellular youthfulness and have a greater capacity to resist

injury and stress, to heal, renew, and regenerate organs and tissues. Thus, compositions and

methods directed at rejuvenating cells, thereby restoring them from an aged, mature state to a a younger, more vital state, have long been sought to treat certain injuries and diseases, as well

as generally reverse and prevent aging in entire organisms.

There

[0004] There are are two two types types of information of information in the in the body: body: digital digital and and analog. analog. DNA DNA is digital is digital

information and the epigenome is analog information. Analog information never lasts as long

as digital, nor can analog information be copied with high fidelity compared to digital

information. This has consequences for how long organisms live and thrive. Aging was once

thought of as a process driven by mutations in the genetic material of a cell. This has largely

PCT/US2019/053545

been abandoned as an explanation. A major cause of aging is now thought to be due to

epigenetic changes that cause cells to transcribe the wrong genes at the wrong time for

optimal function, a process that becomes more dysfunctional over time, leading to diseases,

an inability to heal and eventually to death. The Yamanaka factors (OCT4, SOX2, c-Myc,

and KLF4) have previously been shown to induce pluripotency in vitro (Takahashi et al.,

Cell. 2006 Aug 25;126(4):663-76) and reverse the DNA methlylation clock of aging

(Horvath, Genome Biol. 2013). Nanog and Lin28 can help induce pluoripotency together

with Yamanaka factors. And Tet1, NR5A-2, Sall4, NKX3-1 can replace Oct4 (Gao et al.,

Cell Stem Cell 12, 1-17, April 4, 2013 and Mai et al., Nature Cell Biology 20, 900-908,

2018). Expression of original four transcription factors in transgenic mice, however, induce

teratomas in vivo, along with other acute toxicities like dysplasia in the intestinal epithelium,

that can kill an animal in a few days (Abad et al., Nature. 2013 Oct 17;502(7471):340-5).

Therefore, non-toxic and efficient methods of cellular reprogramming are needed.

SUMMARY OF THE INVENTION

[0005] The cellular aging process has been postulated to be caused by the loss of both

genetic and epigenetic information. While previous studies have hypothesized that aging is

caused primarily by the loss of genetic information (most commonly in the form of genetic

mutations such as substitutions, and deletions in an organism's genome), the compositions

and methods of the present disclosure are informed by the unexpected finding that aging is

primarily driven by a loss in the particular epigenetic information that is established closer to

fertilization and final differentiation of particular cells. Epigenetic information, which

commonly takes the form of covalent modifications to DNA, such as 5-

methylcytosine(5mC), hydroxymethylcytosine (5hmeC), 5-formylcytosine (fC), and 5-

carboxylcytosine (caC) and adenine methylation, and to certain proteins, such as lysine

acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine

ubiquitination and sumoylation of histone proteins, is sometimes referred to as the "analog"

information of the cell. The loss of this analog information can result in dysregulation of vital

cellular processes, such as the processes that maintain cell identity, causing cells to exhibit

traits that are typically associated with aging such as senescence.

[0006] The The methods, methods, compositions, and compositions, and kits kitsofofthe present the disclosure present rejuvenate disclosure cells by rejuvenate cells by

preventing and reversing the cellular causes of aging. Without being bound by a particular

theory, more specifically, the methods, compositions and kits of the present disclosure

rejuvenate cells by restoring epigenetic information that has been lost due to the aging

WO wo 2020/069373 PCT/US2019/053545

process, injury or disease. The methods compositions and kits of the present disclosure

comprise the transcription factors OCT4, SOX2 and KLF4. OCT4, SOX2 and KLF4 are three

of the four "Yamanaka Factors", with the fourth being c-Myc. The Yamanaka Factors have

traditionally been used to reprogram cells to a pluripotent state. However, the induction of

expression of the four transcription factors in transgenic mice resulted in the formation of

which can kill the animal in a few days. Moreover, the fact that the four Yamanaka Factors

are typically used to reprogram cells to a completely pluripotent state, wherein the cell loses

its pre-established cellular identity, can be dangerous for in vivo applications where the

cellular identity of target cells must be maintained for tissue and/or organ integrity. In

contrast, in some embodiments, the methods described herein, allow incomplete

reprogramming and do not result in global changes in demethylation. In some embodiments,

the methods described herein do not require complete de-differentiation of cells. For

example, while expression of OCT4, SOX2, and KLF4 promoted regeneration following

injury in young and old mice and following vincristine-induced injury in human neurons,

expression of OCT4, SOX2, and KLF4 did not induce a global reduction of DNA

methylation (see e.g., FIGs. 45B- 45C).

[0007] In some embodiments, the results disclosed herein suggest that expression of

OCT4, SOX2, and KLF4 can allow diseased cells to revert to a healthier state without

inducing complete reprogramming. Without being bound by a particular theory, the results

disclosed herein suggest that cells maintain a backup epigenome that can be restored using

the methods described herein.

[0008] The The methods, methods, compositions compositions and and kits kits of of the the present present disclosure disclosure are are in in part part informed informed

by the surprising and unexpected discovery that the spatially and temporally specific

induction of OCT4, SOX2, and KLF4 expression in the absence of the induction of c-Myc

expression can rejuvenate a cell without reprogramming the cell to a pluripotent state. Using

inducible promoters, the expression of OCT4, SOX2 and KLF4 can be carefully controlled to

decrease and reverse epigenetic marks associated with aging, increase the epigenetic marks

associated with cellular youthfulness, decrease the expression of aging related proteins,

increase the expression of proteins associated with a youthful cellular state, restore the

balance between euchromatin and heterochromatin, prevent loss of cellular identity, restore

cellular identity, reversing the aging related changes in DNA methylation, thereby

rejuvenating the cell without reprogramming the cell to a pluripotent state.

WO wo 2020/069373 PCT/US2019/053545

[0009] Thus, in various embodiments the methods of the invention rejuvenates a cell by

restoring the cellular identity of the cell by reversing the effects of or preventing of one or

more dysregulated developmental pathways. For example, the methods:

(i) (i) increase the abundance of at least one of histone H2A, histone H2B, histone H3,

histone H4, or any combination thereof in the cell;

(ii) increase the abundance of at least one of CHAFla, CHAF1a, CHAF1b, HP1a, NuRD or HP1, NuRD or any any

combination thereof in the cell;

(iii) increase at least one heterochromatin mark in the cell such as for example

H3K9me3, H3K27me3 or any combination thereof; or decrease one

heterochromatin mark such as H4K20me3 or euchromatin mark H3K4me3; (iv) increase/decrease DNA methylation of at least one age-related CpG site in the cell

towards young level;

(v) increase the abundance of lamin B1 in the cell;

(vi) increase acetylation of histone H3 at lysine 27 (H3K27ac), increase acetylation of

histone H3 at lysine 56 (H3K56ac) or any combination thereof in the cell;

(vii) (vii) decrease decreaseacetylation acetylationofofhistone histoneH3H3atatlysine lysine122 122(H3K122Ac) (H3K122Ac)ororhistone histoneH4H4atat

lysine 16 (H4K16ac), or any combination thereof in the cell

(viii) decrease the abundance of IL6, Ccl2, Ccl20, Apob, p16, LINE-1 repeats, Sat III

repeats, Alu elements, IAP or any combination thereof;

(ix) restores the balance between euchromatin epigenetic marks such as H3K4me3 and

heterochromatin epigenetic marks such as for example H3K9me3 or H3K27me3

(x) induces the formation of euchromatin;

(xi) (xi) restores youthful levels of at least one repressive heterochromatin epigenetic

mark; and/ or

(xii) (xii) restores thethe restores expression of of expression at at least oneone least of of thethe genes recited genes in in recited Table 5 to Table youthful 5 to youthful

levels.

[0010] The present disclosure stems from the unexpected discovery that, in some

embodiments, precise expression of OCT4, SOX2, and KLF4 in the absence of exogenous C- c-

Myc expression can be used to promote reprogramming and tissue regeneration in vivo

without acute toxicity. The expression vectors provided herein, in certain embodiments,

allow for precise control of OCT4, SOX2, and KLF4 (OSK) expression, incorporation into

X 10 viruses (e.g., adeno-associated virus (AAV) at a high viral titer (e.g., more than 2 x 10¹² ¹

particles per preparation, 1 X x 1013 10¹³ particles per mL), reversing aging, treating diseases,

4

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including ocular diseases, and/or tissue regeneration (e.g. optic nerve regeneration) in vivo

following damage.

[0011] As shown in FIG. 14, mice with inducible transgene expression of OCT4, SOX2,

and KLF4 (OSK) died two days after induction of OSK expression, due to generalized

cytological and architectural dysplasia in the intestinal epithelium. A similar finding has been

reported in mice with transgene of OCT4, SOX2, and KLF4 plus c-Myc (Abad et al., Nature.

2013 Oct 17;502(7471):340-5; Ocampo et al., Cell. 2016 Dec 16;167:1719-33). Surprisingly,

in some embodiments as shown in FIG.14, expression of OCT4, SOX2, and KLF4 did not

cause toxicity or cancer in vivo. Continuous expression (e.g., induction by doxycycline

administration) of OCT4, SOX2, and KLF4 through AAV9 delivery (TRE-OSK with UBC-

rtTA4) did not result in teratoma formation in vivo. No teratoma or body weight loss was

detected for three months when AAV9 viruses encoding these three transcription factors were

delivered to the entire body of mice (FIG. 14).

[0012] Accordingly, provided herein, in certain embodiments, are nucleic acids (e.g.,

engineered nucleic acid) capable of inducing expression of OCT4, KLF4, inducing agent,

and/or SOX2 and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia

virus, retrovirus, herpes virus, or AAV) comprising the same. The nucleic acids may encode

OCT4, KLF4, and/or SOX2. The nucleic acids may encode a transcription factor selected

from the group consisting of OCT4; KLF4; SOX2; and any combinations thereof. In certain

embodiments, a nucleic encodes two or more transcription factors selected from the group

consisting of OCT4, KLF4, and SOX2. In certain embodiments, a nucleic acid encodes

OCT4 and SOX2, OCT4 and KLF4. In certain embodiments, a nucleic acid encodes SOX2

and KLF4. In certain embodiments, a nucleic encodes OCT4, KLF4, and SOX2. In certain

embodiments, a nucleic acid encodes four or more transcription factors (e.g., OCT4, SOX2,

KLF4, and another transcription factor). In some embodiments, the present disclosure

provides nucleic acids encoding an inducing agent (e.g., an inducing agent that is capable of

inducing expression of OCT4, KLF4, SOX2, or a combination thereof). In some

embodiments, the nucleic acids encode a Cas9 fusion protein (CRISPR activator) and a guide

RNA sequence targeting a promoter or enhancer at the endogenous locus of OCT4, KLF4,

and/or SOX2. In some embodiments, the nucleic acids encode a Cas9 fusion protein

(CRISPR activator) and a guide RNA sequence targeting a promoter or enhancer at the

endogenous locus of OCT4, SOX2, KLF4, or any combination thereof.

[0013] Aspects of the present disclosure also provide methods of regulating cellular

reprogramming, promoting tissue repair, promoting tissue survival, promoting tissue

PCT/US2019/053545

regeneration, promoting tissue growth, regulating tissue function, promoting organ

regeneration, promoting organ survival, regulating organ function, treating and/or preventing

disease, or any combination thereof. Regulating may comprise inducing cellular

reprogramming, reversing aging, improving tissue function, improving organ function, tissue

repair, tissue survival, tissue regeneration, tissue growth, promoting angiogenesis, reducing

scar formation, reducing the appearance of aging, promoting organ regeneration, promoting

organ survival, altering the taste and quality of agricultural products derived from animals,

treating a disease, or any combination thereof, in vivo or in vitro. The methods may comprise

administering any of the nucleic acids described herein (e.g., DNA and/or RNA), any of the

engineered proteins encoding KLF4, OCT4, an inducing agent, and/or SOX2, any of the

chemical agents activating (e.g., inducing expression of) OCT4, KLF4, an inducing agent,

and/or SOX2, antibodies activating (e.g., inducing expression of) OCT4, KLF4, an inducing

agent, and/or SOX2, and/or any of the recombinant viruses described herein. The methods

may comprise administering any of the nucleic acids described herein (e.g., DNA and/or

RNA), any of the engineered proteins encoding KLF4, SOX2, OCT4, or any combination

thereof, any of the chemical agents activating (e.g., inducing expression of) OCT4; KLF4;

SOX2; or any combination thereof, antibodies activating (e.g., inducing expression of)

OCT4; KLF4; SOX2; or any combination thereof and/or any of the recombinant viruses

described herein. In certain embodiments, the engineered nucleic acids comprise DNA

and/or RNA. The engineered nucleic acid may be an expression vector or not an expression

vector. For example, the engineered nucleic acid may be mRNA or plasmid DNA. In certain

embodiments, the method further comprises administering a nucleic acid (e.g., engineered

nucleic acid) encoding an inducing agent, an engineered protein encoding an inducing agent,

a chemical agent capable of modulating (e.g., activating or inhibiting) the activity of an

inducing agent, and/or a recombinant virus encoding an inducing agent. For example, the

engineered nucleic acid may be mRNA or plasmid DNA.

[0014] One aspect of the present disclosure provide vectors (e.g., expression vectors)

comprising a first nucleic acid (e.g., engineered nucleic acid) encoding OCT4, a second

nucleic acid (e.g., engineered nucleic acid) encoding SOX2, a third nucleic acid (e.g.,

engineered nucleic acid) encoding KLF4, alone or in combination and in the absence of an

exogenous nucleic acid (e.g., engineered nucleic acid) capable of expressing c-Myc. In

certain embodiments, a vector (e.g., expression vector) comprising a first nucleic acid (e.g.,

engineered nucleic acid) encoding OCT4, a second nucleic acid (e.g., engineered nucleic

acid) encoding SOX2, a third nucleic acid (e.g., engineered nucleic acid) encoding KLF4, or

WO wo 2020/069373 PCT/US2019/053545

any combination thereof. In certain embodiments, the first, second, and third nucleic acids

(e.g., engineered nucleic acids) are present on separate expression vectors. In certain

embodiments, two of the first, second, and third nucleic acids (e.g., engineered nucleic acids)

are present on the same expression vector. In some embodiments, all three nucleic acids

(e.g., engineered nucleic acids) are present on the same expression vector. In certain

embodiments, the sequence encoding OCT4 is at least 70% (e.g., at least 75%, 80%, 85%,

90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 2 or 41. In certain embodiments,

the sequence encoding SOX2 is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%,

99%, or 100%) identical to SEQ ID NO: 4 or 43. In certain embodiments, the sequence

encoding KLF4 is at least 70% identical to SEQ ID NO: 6 or 45. In certain embodiments,

OCT4, SOX2, KLF4, or any combination thereof is a human protein. In certain

embodiments, OCT4, SOX2, KLF4, or any combination thereof is a non-human protein (for

example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys);

commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, cats, and/or dogs)

and birds (e.g., commercially relevant birds, such as chickens, ducks, geese, and/or turkeys).

If two or more of OCT4, SOX2, and KLF4 are on one vector, they may be in any order. The

words "first," "second," and "third" are not meant to imply an order of the genes on the

vector.

[0015] An expression vector of the present disclosure may further comprise an inducible

promoter. An expression vector may only have one inducible promoter. In such instances,

the expression of OCT4, SOX2, and KLF4 are under the control of the same inducible

promoter. In some instances, an expression vector comprises more than one inducible

promoter. The inducible promoter may comprise a tetracycline-responsive element (TRE)

(e.g., a TRE3G promoter, a TRE2 promoter, or a P tight promoter), mifepristone-responsive

promoters (e.g., GAL4-Elb promoter), or a coumermycin-responsive). As an example, a

TRE (e.g., TRE3G) promoter may comprise a nucleic acid (e.g., engineered nucleic acid)

sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%)

identical to SEQ ID NO: 7. As an example, a TRE (e.g., TRE2) promoter may comprise a

nucleic acid (e.g., engineered nucleic acid) sequence that is at least 70% (e.g., at least 75%,

80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 23. As an example, a

TRE (e.g., P tight) promoter may comprise a nucleic acid (e.g., engineered nucleic acid)

identical to SEQ ID NO: 24. See, e.g., U.S. Provisional Application, U.S.S.N. 62/738,894,

entitled MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR

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EXPRESSION OF GENES, which was filed on September 28, 2018, under attorney docket

number H0824.70300US00, and the International Patent Application titled MUTANT

REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, under attorney docket number H0824.70300WOOO, H0824.70300WO00, which was filed on the same day as the

instant application, each of which is herein incorporated by reference in its entirety.

[0016] In certain embodiments, an inducing agent is capable of inducing expression of the

first (e.g., OCT4), second (e.g., SOX2), third (e.g., KLF4) nucleic acids (e.g., engineered

nucleic acids), or any combination thereof from the inducible promoter in the presence of a

tetracycline (e.g., doxycycline). In certain embodiments, the inducing agent is reverse

tetracycline-controlled transactivator (rtTA) (e.g., M2-rtTA, rtTA3 or rtTA4). In certain

embodiments, the rtTA is rtTA3 comprising an amino acid sequence that is at least 70% (e.g.,

at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 11. In

certain embodiments, the rtTA is rtTA4 and comprises a sequence that is at least 70% (e.g., at

least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. In

certain embodiments, the rtTA is M2-rtTA and comprises a sequence that is at least 70% %

(e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 15.

[0017] In certain embodiments, an inducing agent is capable of inducing expression of

expression of the first nucleic acid (e.g., engineered nucleic acid) (e.g., OCT4), second

nucleic acid (e.g., engineered nucleic acid) (e.g., SOX2), third nucleic (e.g., KLF4), or any

combination thereof from the inducible promoter in the absence of tetracycline (e.g.,

doxycycline). In certain embodiments, the inducing agent is tetracycline-controlled

transactivator (tTA).

[0018] In certain embodiments, an expression vector of the present disclosure comprises a

constitutive promoter (e.g., CP1, CMV, EF1 alpha, SV40, PGK1, Ubc, human beta actin,

CAG, Ac5, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, and U6 promoter). A constitutive

promoter may be operably linked to nucleic acid (e.g., engineered nucleic acid) sequences

encoding OCT4, KLF4, SOX2, an inducing agent, or a combination thereof. In some

embodiments, an expression vector comprises one constitutive promoter. In some

embodiments, an expression vector comprises more than one constitutive promoter.

[0019] In certain embodiments, an expression vector of the present disclosure comprises

an SV40-derived terminator sequence. In certain embodiments, the SV40-derived sequence

is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to

SEQ ID NO: 8.

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[0020] In certain embodiments, an expression vector of the present disclosure comprises a

separator sequence, which may be useful in producing two separate amino acid sequences

from one transcript. The separator sequence may encode a self-cleaving peptide (e.g., 2A

peptide, including a 2A peptide sequence that is at least 70% (e.g., at least 75%, 80%, 85%,

90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 9). In certain embodiments, the

separator sequence is an Internal Ribosome Entry Site (IRES).

[0021] In certain embodiments, the expression vector is a viral vector (e.g., a lentiviral, a

retroviral, or an adeno-associated virus (AAV) vector) (e.g., FIGS. 2-3). An AAV vector of

the present disclosure generally comprises inverted terminal repeats (ITRs) flanking a

transgene of interest (e.g., a nucleic acid (e.g., engineered nucleic acid) encoding OCT4,

SOX2, KLF4, an inducing agent, or a combination thereof). In some embodiments, the

distance between two inverted terminal repeats is less than 5.0 kilobases (kb) (e.g., less than

4.9 kb, less than 4.8 kb, less than 4.7 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb,

less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4 kb, less than 3.5 kb, less than 3

kb, less than 2.5 kb, less than 2 kb, less than 1.5 kb, less than 1 kb, or less than 0.5 kb).

[0022] In certain embodiments, an expression vector (e.g., an expression vector encoding

OCT4, KLF4, SOX2, an inducing agent, or a combination thereof) of the present disclosure

may further comprise a selection agent (e.g., an antibiotic, including blasticidin, geneticin,

hygromycin B, mycophenolic acid, puromycin, zeocin, actinomycin D, ampicillin,

carbenicillin, kanamycin, and neomycin) and/or detectable marker (e.g., GFP, RFP,

luciferase, CFP, mCherry, DsRed2FP, mKate, biotin, FLAG-tag, HA-tag, His-tag, Myc-tag,

V5-tag, etc.).

[0023] In some embodiments, the expression vector (e.g., viral vector) encoding OCT4,

KLF4, and SOX2 comprises the sequence provided in SEQ ID NO: 16, SEQ ID NO: 105, or

SEQ ID NO: 121. In some embodiments, the expression vector encoding OCT4, KLF4, and

SOX2 comprise the elements depicted in FIG. 2, FIG. 3, FIGS. 4A-4AL, FIGS. 5A-5D, or a

combination thereof. Viral vectors include adeno-associated virus (AAV) vectors, retroviral

vectors, lentiviral vectors, and herpes viral vectors.

[0024] In another aspect, the present disclosure provides recombinant viruses (e.g.,

lentivirus, adenovirus, retrovirus, herpes virus, alphavirus, vaccinia virus or adeno-associated

virus (AAV)) comprising any of the expression vectors described herein. In certain

embodiments, a recombinant virus encodes a transcription factor selected from OCT4; KLF4;

SOX2; and any combinations thereof. In certain embodiments, a recombinant virus encodes

two or more transcription factors selected from the group consisting of OCT4, KLF4, and

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SOX2. In certain embodiments, a recombinant virus encodes OCT4 and SOX2, OCT4 and

KLF4, OCT4, KLF4, and SOX2, or SOX2 and KLF4. In certain embodiments, a

recombinant virus encodes OCT4, KLF4, and SOX2. In certain embodiments, a four or more

transcription factors encodes four or more transcription factors (e.g., OCT4, SOX2, KLF4,

and another transcription factor).

[0025] In yet another aspect, the present disclosure provides methods of regulating (e.g.,

inducing) cellular reprogramming, tissue repair, tissue regeneration, organ regeneration,

reversing aging, or any combination thereof comprising administering to a cell a first nucleic

acid (e.g., engineered nucleic acid) encoding OCT4, a second nucleic acid (e.g., engineered

nucleic acid) encoding SOX2, and a third nucleic acid (e.g., engineered nucleic acid)

encoding KLF4 in the absence of an exogenous nucleic acid (e.g., engineered nucleic acid)

capable of expressing c-Myc. In certain embodiments, the first nucleic acid (e.g., engineered

nucleic acid) encoding OCT4, the second nucleic acid (e.g., engineered nucleic acid)

encoding SOX2, and the third nucleic acid (e.g., engineered nucleic acid) encoding KLF4 is

administered to a subject. The subject may be human or non-human. Non-human subjects

include, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys);

In certain embodiments, the three nucleic acids (e.g., engineered nucleic acids) are

administered simultaneously. In certain embodiments, the three nucleic acids (e.g.,

engineered nucleic acids) are administered simultaneously on the same vector.

[0026] In yet another aspect, the present disclosure provides methods of regulating (e.g.,

nucleic acid) encoding SOX2, a third nucleic acid (e.g., engineered nucleic acid) encoding

KLF4, KLF4, or orany anycombination thereof. combination In certain thereof. embodiments, In certain the firstthe embodiments, nucleic firstacid (e.g.,acid (e.g., nucleic

engineered nucleic acid) encoding OCT4, the second nucleic acid (e.g., engineered nucleic

acid) encoding SOX2, the third nucleic acid (e.g., engineered nucleic acid) encoding KLF4,

or any combination thereof is administered to a subject.

[0027] The expression vector comprising one or more of the first, second, and third

nucleic acids (e.g., engineered nucleic acids) may be any of the expression vectors described

above and herein. In some embodiments, the first nucleic acid, the second nucleic acid, the

third nucleic acid, or any combination thereof are present on separate expression vectors. In

WO wo 2020/069373 PCT/US2019/053545

certain embodiments, two of the first nucleic acid, the second nucleic acid, the third nucleic

acid, or any combination thereof are present on the same expression vector. In certain

embodiments, all three nucleic acids (e.g., engineered nucleic acids) are present on the same

expression vector. In certain embodiments, at least two of the first, second, or third nucleic

acids (e.g., engineered nucleic acids) are operably linked to the same promoter. In certain

embodiments, all three of the first, second, and third nucleic acids (e.g., engineered nucleic

acids) are operably linked to the same promoter.

[0028] In some embodiments, the expression vector (e.g., viral expression vector,

including lentiviral, retroviral, adeno-associated viral vectors) comprises an inducible

promoter (e.g., a promoter comprising a tetracycline-responsive element (TRE) including a

TRE3G sequence, a TRE2 sequence, or a P tight sequence), and the method further

comprises administering an inducing agent (e.g., a chemical agent, a nucleic acid (e.g.,

engineered nucleic acid) (e.g., nucleic acid (e.g., engineered nucleic acid) encoding an

inducing agent), a protein, light, or temperature). In some embodiments, a pH is used to

induce expression of a nucleic acid operably linked to a promoter. In certain embodiments, a

chemical agent capable of modulating the activity of an inducing agent is tetracycline (e.g.,

doxycycline). As a non-limiting example, tetracycline-controlled transactivator (tTA) is an

inducing agent whose activity is inhibited by tetracycline. As a non-limiting example,

reverse tetracycline-controlled transactivator (rtTA) is an inducing agent whose activity is

activated by tetracycline. The inducing agent (e.g., rtTA or tTA) may be encoded by a fourth

nucleic acid (e.g., engineered nucleic acid) that is administered nucleic acid. In certain

embodiments, the inducing agent (e.g., a chemical agent, a nucleic acid (e.g., engineered

nucleic acid) (e.g., a nucleic acid comprising RNA and/or DNA encoding an inducing agent),

a protein, light, a particular pH, or temperature) is introduced simultaneously with the nucleic

acids (e.g., engineered nucleic acids) encoding OCT4, SOX2, and KLF4. In certain

acids (e.g., engineered nucleic acids) encoding one or more (e.g., two or more or three or

more) transcription factors selected from OCT4; SOX2; KLF4; and any combinations

thereof. A promoter (e.g., constitutive promoter, including CAG and Ubc, or an inducible

promoter) may be operably linked to the nucleic acid (e.g., engineered nucleic acid) encoding

the inducing agent. In certain embodiments, the promoter operably linked to the nucleic acid

(e.g., engineered nucleic acid) encoding the inducing agent is a tissue-specific promoter.

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[0029] In certain In certainembodiments, embodiments,the the nucleic acid (e.g., nucleic engineered acid (e.g., nucleic acid) engineered encoding nucleic acid) encoding

the inducing agent is present on the same expression vector as at least one of the nucleic acids

(e.g., engineered nucleic acids) encoding OCT4, SOX2, KLF4, or a combination thereof. In

certain embodiments, the nucleic acid (e.g., engineered nucleic acid) encoding the inducing

agent is present on a separate expression vector from the nucleic acid (e.g., engineered

nucleic acid) encoding OCT4, the nucleic acid (e.g., engineered nucleic acid) SOX2, and the

nucleic acid (e.g., engineered nucleic acid) encoding KLF4. In certain embodiments, the

nucleic acids (e.g., engineered nucleic acids) encoding OCT4, SOX2, and KLF4 are present

on a first expression vector, and the fourth nucleic acid (e.g., engineered nucleic acid) is

present on a second expression vector.

[0030] In some embodiments, a nucleic acid encoding OCT4, SOX2, KLF4, and/or an

inducing agent is not present on a viral vector. In some embodiments, a nucleic acid

encoding one or more (e.g., two or more or three or more) transcription factors selected from

the group consisting of OCT4; SOX2; KLF4; and any combinations thereof is not present on

a viral vector. In some embodiments, the nucleic acid is delivered without a viral vector. In

some embodiments, delivery of the nucleic acid that is not on a viral vector comprises

administration of a naked nucleic acid, electroporation, use of a nanoparticle, and/or use of a

liposome.

[0031] The expression vectors may be viral vectors (e.g., lentivirus vectors, adenovirus

vectors, retrovirus vectors, herpes virus vectors, alphavirus, vaccinia virus, or AAV vectors).

For example, the first expression vector encoding OCT4, SOX2, and KLF4 may comprise the

nucleic acid (e.g., engineered nucleic acid) sequence set forth in SEQ ID NO: 16. In some

embodiments, the expression vector encoding an inducing agent comprises the sequence

provided in SEQ ID NO: 17 (e.g., FIG. 12), SEQ ID NO: 31 (e.g., FIG. 18), or SEQ ID NO:

32 (e.g., FIG. 19).

[0032] In certain embodiments, the fourth nucleic acid (e.g., engineered nucleic acid)

encoding the inducing agent further comprises an SV-40-derived terminator sequence,

including a sequence that is at least 70% identical to SEQ ID NO: 8.

[0033] In certain embodiments, the inducing agent is capable of inducing expression from

the inducible promoter in the presence of tetracycline (e.g., doxycycline). In certain

embodiments, the inducing agent is rtTA (e.g., rtTA3, including rtTA3 with a sequence that

is at least 70% identical to SEQ ID NO: 11, and rtTA4, including rtTA4 with a sequence that

is at least 70% identical to SEQ ID NO: 13). In certain embodiments, the method further

comprises administering tetracycline (e.g., doxycycline) to the cell, tissue, or subject. In

WO wo 2020/069373 PCT/US2019/053545

certain embodiments, the method comprises removing tetracycline (e.g., doxycycline) from

the cell, tissue, or subject.

[0034] In certain embodiments, the inducing agent is capable of inducing expression from

the inducible promoter in the absence of tetracycline (e.g., doxycycline). In certain

embodiments, the inducing agent is a tetracycline transactivator (tTA). Without being bound

by a particular theory, tetracycline (e.g., doxycycline) may bind to the tTA and prevent tTA

from binding its cognate promoter (e.g., a promoter comprising a tetracycline response

element (TRE)) and driving expression of an operably linked nucleic acid. Without being

bound by a particular theory, a nucleic acid (e.g., engineered nucleic acid) encoding an

inducing agent may not be on the same vector as any of the nucleic acids (e.g., engineered

nucleic acids) encoding OCT4, KLF4, and SOX2 to reduce the size of a viral vector and

improve viral titer.

[0035] In certain embodiments, one or more expression vectors (e.g., AAV comprising an

expression vector) is administered to a cell, tissue, or a subject in need thereof. The subject

may have an injury or condition, is suspected of having a condition or injury, or is at risk for

a a condition conditionoror injury. Without injury. beingbeing Without bound bound by a particular theory, expression by a particular of the theory, expression of the

transcription factors OCT4, SOX2, and KLF4 induces cellular reprogramming. In some

embodiments, when the nucleic acid (e.g., engineered nucleic acid) encoding OCT4, SOX2,

KLF4, or a combination thereof is operably linked to an inducible promoter, administration

of an inducing agent (e.g., chemical, a protein, a nucleic acid (e.g., engineered nucleic acid)

(e.g., a nucleic acid (e.g., engineered nucleic acid) encoding an inducing agent) under the

appropriate conditions (e.g., in the presence or absence of tetracycline). In certain

embodiments, an inducing agent (e.g., rtTA) is capable of binding a promoter and driving

expression of an operably linked nucleic acid (e.g., engineered nucleic acid) only when the

inducing agent is bound to tetracycline. In certain embodiments, an inducing agent (e.g.,

tTA) cannot bind a promoter and drive expression of an operably linked nucleic acid (e.g.,

engineered nucleic acid) when the inducing agent is bound to tetracycline. The condition

may be an ocular disease, (e.g., a retinal disease, a corneal disease, or any disease affecting

the eye), cancer, aging, an age-related disease, injury, or a neurodegenerative disease. In

certain embodiments, the cell or tissue is from eye, ear, nose, mouth including gum and roots

of teeth, bone, lung, breast, udder, pancreas, stomach, oesophagus, muscle including cardiac

muscle, liver, blood vessel, skin including hair, heart, brain, nerve tissue, kidney, testis,

prostate, penis, cloaca, fin, ovary, or intestine.

WO wo 2020/069373 PCT/US2019/053545

[0036] In certain embodiments, the tissue is damaged (e.g., due to an injury, an accident,

or an iatrogenic injury) and/or is aged tissue. In certain embodiments, the tissue may be

considered healthy but suboptimal for performance or survival in current or future conditions

(e.g., in agriculture or adverse conditions including toxic therapies, sun exposure, or travel

outside the earth's atmosphere).

[0037] In certain embodiments, the method comprises further comprises regulation of a

biological process. In some embodiments, the methods described herein comprise regulating

any biological process, including, cellular reprogramming, tissue repair, tissue survival, tissue

regeneration, tissue growth, tissue function, organ regeneration, organ survival, organ

function, or any combination thereof. In some embodiments, the methods comprise inducing

cellular reprogramming, reversing aging, improving tissue function, improving organ

function, promoting tissue repair, promoting tissue survival, promoting tissue regeneration,

promoting tissue growth, promoting angiogenesis, reducing scar formation, reducing the

appearance of aging including alopecia, hair thining, hair greying, sagging skin, and skin

wrinkles, promoting organ regeneration, promoting organ survival, altering the taste and

quality of agricultural products derived from animals, treating a disease, or any combination

thereof, in vivo or in vitro. For example, the method may induce cellular reprogramming, cell

survival, organ regeneration, tissue regeneration, or a combination thereof. In certain

embodiments, the method comprises inducing and then stopping cellular reprogramming, cell

survival, tissue regeneration, organ regeneration, aging, or a combination thereof. In certain

embodiments, the method reverses aging of a cell, tissue, organ, or subject. In some

embodiments, the method does not induce teratoma formation. In some embodiments, the

method does not induce unwanted cell proliferation. In some embodiments, the method does

not induce malignant cell growth. In some embodiments, the method does not induce cancer.

In some embodiments, the method does not induce tumor growth or tumor formation. In

some embodiments, the method does not induce glaucoma.

[0038] In some embodiments, a method described herein reverses the epigenetic clock of a

cell, cell, aa tissue, tissue,an an organ, organ, a subject, a subject, or anyorcombination any combination thereof.thereof. In some embodiments, In some embodiments, the the

epigenetic clock is determined using a DNA methylation-based (DNAm) age estimator. In

some embodiments, the method alters the expression of one or more genes associated with

ageing. In some embodiments, the method reduces expression of one or more genes

associated with ageing. In some embodiments, the method alters the expression of one or

more genes associated with ageing. In some embodiments, the one or more genes is one or

more sensory genes.

WO wo 2020/069373 PCT/US2019/053545

[0039] In some embodiments, the method reduces expression of one or more genes

associated with ageing. In some embodiments, the method reduces expression of

0610040J01Rik, 1700080N15Rik, 2900064F13Rik, 4833417C18Rik, 4921522P10Rik,

4930447C04Rik, 4930488N15Rik, Ace, Ackr1, Acot10, Acvrl, Acvr1, Adamts17, Adralb,

AI504432, Best3, Boc, Cadm3, Cand2, Ccl9, Cd14, Cd36, Cfh, Chrm3, Chrna4, Cntn4,

Cracr2b, Cryaa, CT573017.2, Cyp26a1, Cyp27a1, D330050G23Rik, D930007P13Rik, Ddo,

Dgkg, Dlk2, Dnajal-ps, Dnaja1-ps, Drd2, Dsel, Dytn, Ecscr, Edn1, Ednrb, Efemp1, Elfn2, Epha10,

Ephx1, Erbb4, Fam20a, Fbxw21, Ffar4, Flt4, Fmod, Foxp4, Fzd7, Gabrd, Galnt15, Galnt18,

Gfra2, Ggt1, Gm10416, Gm14964, Gm17634, Gm2065, Gm32352, Gm33172, Gm34280,

Gm35853, Gm36298, Gm36356, Gm36937, Gm3898, Gm42303, Gm42484, Gm42537,

Gm42743, Gm43151, Gm43843, Gm44545, Gm44722, Gm45516, Gm45532, Gm47494,

Gm47982, Gm47989, Gm48398, Gm48495, Gm48593, Gm48958, Gm49089, Gm49326, Gm49331, Gm49760, Gm5796, Gm6374, Gm7276, Gm8237, Gm9796, Gm9954, Gpr75,

Gprc5c, Grid2ip, Gsg112, Hapln4, Hcn3, Hcn4, Hhatl, Hs6st2, Htr3a, Illrap, Il1rapl2, Inkal, Inka1,

Kbtbd12, Kcnj11, Kcnk4, Kdelc2, Klhl33, Lamc3, Lilra5, Lman11, Lrfn2, Lrrc38, Lrrn4cl,

Ltc4s, Manscl, Mansc1, Mir344c, Msr1, Mycbpap, Myoc, Ngfr, Nipal2, Olfr1372-ps1, Otop3, P2rx5,

P2ry12, P4ha2, Pcdha12, Pcdha2, Pcdhac2, Pcdhb18, Pcdhb5, Pcsk2os1, Pcsk6, Perp, Pkp1,

Plxna4, Prickle2, Qsox1, Rapgef4os2, Rbp4, Rcn3, Sec1415, Sel113, Serpinh1, Sgpp2,

Shisa6, Siah3, Siglech, Slc12a4, Slc24a2, Slc2a5, Slc4a4, Slitrk3, Smagp, Smoc2, Speer4b,

Spon2, Sstr2, Sstr3, St3gal3, Stcl, Stc1, Stc2, Syndig1, Syt10, Thsd7a, Tlr8, Tmem132a,

Tmem132d, Tmem200a, Tmem44, Trpc4, Trpv4, Unc5b, Vgf, Vmn1r90, Vwc21, Wfikkn2,

Wnt11, Wnt6, Zeb2os, Zfp608, Zfp976, or any combination thereof. In some embodiments,

the method reduces expression of Ace, Kcnk4, Lamc3, Edn1, Syt10, Ngfr, Gprc5c, Cd36,

Chrna4, Ednrb, Drd2, or a combination thereof.

[0040] In some embodiments, the method increases expression of one or more genes

associated with ageing. In some embodiments, the method increases expression of

1700031P21Rik, 1810053B23Rik, 2900045O20Rik, 2900060B14Rik, 4921504E06Rik,

4930402F11Rik, 4930453C13Rik, 4930455B14Rik, 4930500H12Rik, 4930549P19Rik,

4930555B11Rik, 4930556J02Rik, 4932442E05Rik, 4933431K23Rik, 4933438K21Rik,

6720475M21Rik, 9830132P13Rik, A430010J10Rik, A530064D06Rik, A530065N20Rik,

Abcb5, Abhd17c, AC116759.2, AC131705.1, AC166779.3, Acot12, Adig, Akr1cl, Ankrd1,

Asb15, Atp2c2, AU018091, AW822073, Btnl10, Btn110, C130093G08Rik, C730027H18Rik,

Ccdc162, Chil6, Col26a1, Corin, Crls1, Cybrd1, Cyp2d12, Cyp7a1, D830005E20Rik, Dlx3,

Dnah14, Dsc3, Dthd1, Eid2, Eps811, EU599041, Fam90a1a, Fancf, Fau-ps2, Fezf1, Gja5,

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

Gm10248, Gm10513, Gm10635, Gm10638, Gm10718, Gm10722, Gm10800, Gm10801,

Gm11228, Gm11251, Gm11264, Gm11337, Gm11368, Gm11485, Gm11693, Gm12793,

Gm13050, Gm13066, Gm13323, Gm13339, Gm13346, Gm13857, Gm14387, Gm14770,

Gm15638, Gm16072, Gm16161, Gm16181, Gm17200, Gm17791, Gm18025, Gm18757,

Gm18795, Gm18848, Gm19719, Gm20121, Gm20356, Gm2093, Gm21738, Gm21940,

Gm22933, Gm24000, Gm24119, Gm25394, Gm26555, Gm27047, Gm28262, Gm28530,

Gm29295, Gm29825, Gm29844, Gm3081, Gm32051, Gm32122, Gm33056, Gm33680,

Gm34354, Gm34643, Gm3551, Gm36660, Gm36948, Gm37052, Gm37142, Gm37262,

Gm37535, Gm37569, Gm37589, Gm37647, Gm37648, Gm37762, Gm38058, Gm38069,

Gm38137, Gm38218, Gm39139, Gm42535, Gm42680, Gm42895, Gm42994, Gm43027,

Gm43158, Gm43288, Gm43366, Gm44044, Gm44081, Gm44187, Gm44280, Gm44535,

Gm45338, Gm45644, Gm45740, Gm46555, Gm46565, Gm4742, Gm47485, Gm47853,

Gm47992, Gm48225, Gm48314, Gm48383, Gm48673, Gm48804, Gm48832, Gm4994,

Gm5487, Gm5724, Gm595, Gm6012, Gm6024, Gm7669, Gm7730, Gm8043, Gm8953, Gm9348, Gm9369, Gm9495, H2al2a, Ido2, Igfbp1, Kif7, Klhl31, Lrrc31, Mc5r, Mgam,

Msh4, Mucl2, Mugl, Mug1, Mybl2, Myh15, Nek10, Neurod6, Nr1h5, Olfr1042, Olfr1043,

Olfr1082, Olfr1090, Olfr1124, Olfr1167, Olfr1205, Olfr1206, Olfr1223, Olfr1263, Olfr1264,

Olfr1269, Olfr127, Olfr1291-ps1, Olfr1406, Olfr1469, Olfr215, Olfr273, Olfr328, Olfr355,

Olfr372, Olfr390, Olfr427, Olfr456, Olfr466, Olfr481, Olfr522, Olfr6, Olfr601, Olfr603,

Olfr706, Olfr727, Olfr728, Olfr741, Olfr801, Olfr812, Olfr816, Olfr822, Olfr860, Olfr890,

Olfr923, Olfr943, Otogl, Pi15, Pkhd1, Pkhd111, Platr6, Pou3f4, Prr9, Pvalb, Rhag, Sav1,

Serpinb9b, Skint1, Skint3, Skint5, Slc10a5, Slc6a4, Smok2a, Tcaf3, Tomm201, Trcg1, Trdn,

Ugt1a6a, Usp171a, Usp17la, Vmn1r178, Vmn1r179, Vmn1r33, Vmn1r74, Vmn1r87, Vmn2r102,

Vmn2r113, Vmn2r17, Vmn2r52, Vmn2r66, Vmn2r68, Vmn2r76, Vmn2r78, Wnt16, or any

combination thereof. In some embodiments, the method increases expression of Olfr816,

Olfr812, Olfr1264, Olfr727, Olfr923, Olfr1090, Olfr328, Olfr1124, Olfr522, Olfr1082,

Olfr1206, Olfr1167, Olfr706, Olfr6, Pou3f4, Olfr603, Olfr127, Olfr1263, Olfr1269,

Olfr1205, Olfr390, Olfr601, Olfr860, Olfr215, Olfr741, Olfr1469, Olfr355, Olfr481, Olfr456,

Olfr1042, Olfr728, Olfr372, Olfr801, Olfr1223, Olfr822, Otogl, Olfr943, Olfr1406, Olfr273,

Olfr466, Olfr1043, Olfr427, Olfr890, Rbp4, or any combination thereof.

[0041] Further aspects of the disclosure relate to methods of reprogramming comprising

rejuvenating rejuvenating the the epigenetic epigenetic clock clock of of aa cell, cell, tissue, tissue, organ, organ, subject, subject, or or any any combination combination thereof. thereof.

[0042] Further aspects Further of the aspects disclosure of the relate disclosure to methods relate of reprogramming to methods comprising of reprogramming comprising

altering the expression of one or more genes associated with ageing.

WO wo 2020/069373 PCT/US2019/053545

[0043] Further aspects of the disclosure relate to methods comprising resetting the

transcriptional profile of an old cell, an old organ, an old tissue, and/or any combination

thereof in vitro.

[0044] Further aspects of the disclosure relate to methods comprising resetting the

transcriptional profile of an old cell, an old organ, an old tissue, an old subject and/or any

combination thereof in vivo.

[0045] Further aspects of the disclosure relate to methods of transdifferentiating cells, cells.

[0046] Another aspect of the present disclosure provides engineered cells generated by

any of the methods described herein. The methods described herein may be useful in the

production of any engineered cell, including induced pluripotent stem cells. The engineered

cells of the present disclosure may be produced ex vivo and the methods may further

comprise generating an engineered tissue or engineered organ. In some embodiments, the

methods of the present disclosure comprise administering an engineered cell, engineered

tissue, and/or engineered organ of the present disclosure to a subject in need thereof. In some

embodiments, the method further comprises treating a disease.

[0047] Aspects of the present disclosure also provide compositions comprising any of the

nucleic acids (e.g., engineered nucleic acid) capable of inducing OCT4, KLF4, inducing

agent, and/or SOX2 expression (e.g., expression vector), any of the engineered proteins

described herein, any of the chemical agents activating (e.g., inducing expression of) OCT4,

KLF4, an inducing agent, and/or SOX2, any of the antibodies activating (e.g., inducing

expression of) OCT4, KLF4, an inducing agent, and/or SOX2, and/or any of the recombinant

viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or

AAV) described herein, alone, or in combination. In some embodiments, the pharmaceutical

compositions of the present disclosure further comprise a pharmaceutically acceptable

carrier. In some embodiments, the pharmaceutical composition further comprises a second

engineered nucleic acid (e.g., engineered nucleic acid) (e.g., expression vector including viral

vector) encoding an inducing agent (e.g., rtTA or tTA).

[0048] Aspects of the present disclosure also provide compositions comprising any of the

nucleic acids (e.g., engineered nucleic acid) acids capable of inducing OCT4, KLF4, , and/or and/or

SOX2 (e.g., expression vector, including an inducible expression vector), any of the

engineered proteins described herein, any of the chemical agents capable of activating (e.g.,

inducing expression of) OCT4, KLF4, and/or SOX2, any of the antibodies capable of

activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2, and/or any of the

recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes

17

WO wo 2020/069373 PCT/US2019/053545

virus, or AAV) described herein, alone or in combination. In some embodiments, the

composition further comprises a nucleic acid (e.g., engineered nucleic acid) encoding an

inducing agent, an engineered protein encoding an inducing agent, a chemical agent capable

of modulating (e.g., activating or inhibiting) the activity of an inducing agent, and/or a

recombinant virus encoding an inducing agent. In some embodiments, the pharmaceutical

vector) encoding an inducing agent (e.g., rtTA or tTA).

[0049] Aspects of the present disclosure also provide compositions comprising any of the

nucleic acids (e.g., engineered nucleic acid) (e.g., expression vector) capable of inducing

expression of one or more transcription factors selected from OCT4; SOX2; KLF4; and any

combination thereof, any of the engineered proteins described herein, any of the chemical

agents activating (e.g., inducing expression of) one or more transcription factors selected

from the group consisting of OCT4; SOX2; KLF4; and any combinations thereof, any of the

antibodies activating (e.g., inducing expression of) one or more transcription factors selected

from the group consisting of OCT4; SOX2; KLF4; and any combinations thereof, and/or any

of the recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus,

herpes virus, or AAV) described herein, alone or in combination. In certain embodiments, a

composition comprises any of the nucleic acids (e.g., engineered nucleic acid) (e.g.,

expression vector) capable of inducing expression of two or more transcription factors

selected from OCT4; SOX2; KLF4; and any combination thereof, any of the engineered

proteins described herein, any of the chemical agents activating (e.g., inducing expression of)

two or more transcription selected from the group consisting of OCT4; SOX2; KLF4; and

any combinations thereof, any of the antibodies activating (e.g., inducing expression of) two

or more transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and

any combinations thereof, and/or any of the recombinant viruses (e.g., lentivirus, adenovirus,

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) described herein, alone or in

combination. The two or more transcription factors may comprise OCT4 and SOX2, OCT4

and KLF4, OCT4, KLF4, and SOX2, or SOX2 and KLF4. In certain embodiments, a

expression vector) capable of inducing expression of three or more transcription factors

selected from OCT4, SOX2, KLF4, and combinations thereof expression, any of the

engineered proteins described herein, any of the chemical agents activating (e.g., inducing

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WO wo 2020/069373 PCT/US2019/053545

expression of) three or more transcription selected from the group consisting of OCT4;

SOX2; KLF4; and any combinations thereof, any of the antibodies activating (e.g., inducing

expression of) three or more transcription factors selected from the group consisting of

OCT4; SOX2; KLF4; and any combinations thereof, and/or any of the recombinant viruses

(e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV)

described herein, alone or in combination. In certain embodiments, the three or more

transcription factors may comprise OCT4, SOX2, and KLF4. In some embodiments, a

pharmaceutical composition further comprises a nucleic acid (e.g., engineered nucleic acid)

(e.g., expression vector) encoding an inducing agent (e.g., rtTA or tTA), any of the

engineered proteins encoding an inducing agent, any of the chemical agents capable of

activating (e.g., inducing expression of) an inducing agent, and/or any of the recombinant

AAV) encoding an inducing agent. In some embodiments, the pharmaceutical compositions

of the present disclosure further comprise a pharmaceutically acceptable carrier.

[0050] In yet another aspect, the present disclosure provides kits comprising any of the

AAV) described herein.

[0051] In yet another aspect, the present disclosure provides kits comprising any of the

nucleic acids (e.g., engineered nucleic acid) acids capable of inducing OCT4, KLF4, and/or

SOX2 expression (e.g., expression vector), any of the engineered proteins described herein,

any of the chemical agents activating (e.g., inducing expression of) OCT4, KLF4, and/or

SOX2, any of the antibodies activating (e.g., inducing expression of) OCT4, KLF4, and/or

SOX2, and/or any of the recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

vaccinia virus, retrovirus, herpes virus, or AAV) described herein. In some embodiments,

the kit further comprises a nucleic acid (e.g., engineered nucleic acid) encoding an inducing

agent, an engineered protein encoding an inducing agent, a chemical agent capable of

modulating (e.g., activating or inhibiting) the activity of an inducing agent, and/or a

recombinant virus encoding an inducing agent.

WO wo 2020/069373 PCT/US2019/053545

[0052] In yet another aspect, the present disclosure provides kits comprising any of the

expression of one or more transcription factors selected from the group consisting of OCT4;

SOX2; KLF4; and any combinations thereof thereof,,any anyof ofthe theengineered engineeredproteins proteinsdescribed described

herein, any of the chemical agents activating (e.g., inducing expression of) one or more

transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and any

combinations thereof, any of the antibodies activating (e.g., inducing expression of) OCT4;

SOX2; KLF4; and any combinations thereof, and/or any of the recombinant viruses (e.g.,

lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) described

herein. In certain embodiments, a kit further comprises a nucleic acid (e.g., engineered

nucleic acid) (e.g., expression vector) encoding an inducing agent (e.g., rtTA or tTA), any of

the engineered proteins encoding an inducing agent, any of the chemical agents capable of

AAV) encoding an inducing agent.

[0053] The details of one or more embodiments of the invention are set forth herein. Other

features, objects, and advantages of the invention will be apparent from the Detailed

Description, Examples, Figures, and Claims.

[0054] References cited in this application are incorporated herein by reference.

DEFINITIONS

[0055] Definitions of specific terms are described in more detail below. The disclosure is

not intended to be limited in any manner by the exemplary listing of substituents described

herein.

[0056] "AAV" or "adeno-associated virus" is a nonenveloped virus that is capable of

carrying and delivering nucleic acids (e.g., engineered nucleic acids) (e.g., nucleic acids (e.g.,

engineered nucleic acids) encoding OCT4; KLF4; SOX2; or any combination thereof) and

belongs to the genus Dependoparvovirus. In some instances, an AAV is capable of

delivering a nucleic acid encoding an inducing agent. In general, AAV does not integrate

into the genome. The tissue-specific targeting capabilities of AAV is often determined by the

AAV capsid serotype (see, e.g., Table 1 below for examples of AAV serotypes and their

utility in tissue-specific delivery). Non-limiting serotypes of AAV include AAV1, AAV2,

AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. In certain embodiments, the AAV serotype is a variant of AAV9 (e.g., AAV PHP.b).

WO wo 2020/069373 PCT/US2019/053545

[0057] A "recombinant virus" is a virus (e.g., lentivirus, adenovirus, retrovirus, herpes

virus, alphavirus, vaccinia virus or adeno-associated virus (AAV))) that has been isolated

from its natural environment (e.g., from a host cell, tissue, or a subject) or is artificially

produced.

[0058] The The termterm "AAV"AAV vector" vector" as used as used herein herein is aisnucleic a nucleic acidacid (e.g., (e.g., engineered engineered nucleic nucleic

acid) that comprises AAV inverted terminal repeats (ITRs) flanking an expression cassette

(e.g., an expression cassette comprising a nucleic acid (e.g., engineered nucleic acid)

encoding OCT4, KLF4, and SOX2, each alone or in combination, or an expression cassette

encoding rtTA or tTA). An AAV vector may further comprise a promoter sequence.

[0059] The The terms terms "administer," "administer," "administering," "administering," or "administration," or "administration," as used as used herein herein refers refers

to introduction of any of the compositions described herein, any of the nucleic acids (e.g.,

engineered nucleic acid) capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g.,

expression vector), any of the nucleic acids (e.g., engineered nucleic acid) (e.g., expression

vector) capable of inducing expression of one or more transcription factors selected from the

group consisting of OCT4; KLF4; SOX2; and any combinations thereof, any of the

expression of) OCT4, KLF4, and/or SOX2, any of the chemical agents activating (e.g.,

inducing expression of) one or more transcription factors selected from OCT4; KLF4; SOX2;

and any combinations thereof, any of the antibodies activating (e.g., inducing expression of)

OCT4, KLF4, and/or SOX2, any of the antibodies activating (e.g., inducing expression of)

one or more transcription factors selected from OCT4; KLF4; SOX2; and any combinations

thereof, and/or any of the recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

vaccinia virus, retrovirus, herpes virus, or AAV) described herein, alone, or in combination

to any cell, tissue, organ, and/or subject. In some embodiments, a nucleic acid (e.g.,

engineered nucleic acid) encoding an inducing agent, an engineered protein encoding an

inducing agent, a chemical agent capable of modulating (e.g., activating or inhibiting) the

activity of an inducing agent, and/or a recombinant virus encoding an inducing agent is also

administered administered to to the the cell, cell, tissue, tissue, organ organ and/or and/or subject. subject. Any Any of of the the compositions compositions described described

herein, comprising any of the nucleic acids (e.g., engineered nucleic acid) capable of

inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), comprising any of

the nucleic acids (e.g., engineered nucleic acid) (e.g., expression vector) capable of inducing

expression of one or more transcription factors selected from OCT4; KLF4; SOX2; and any

combinations thereof, any of the engineered proteins described herein, any of the chemical

agents activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2, any of the

21

WO wo 2020/069373 PCT/US2019/053545

engineered proteins encoding OCT4, SOX2, KLF4, or any combinations thereof, any of the

chemical agents activating (e.g., inducing expression of) OCT4; KLF4; SOX2; or any

combination thereof, any of the antibodies activating (e.g., inducing expression of) OCT4,

KLF4, and/or SOX2, any of the antibodies activating (e.g., inducing expression of) OCT4;

KLF4; SOX2; or any combination thereof, and/or any of the recombinant viruses (e.g.,

herein, alone, or in combination may be administered intravenously, intradermally,

intraarterially, intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly,

intrapleurally, intranasally, intravitreally, intravaginally, intrarectally, topically,

intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival,

intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally,

topically, locally, systemically, injection, infusion, continuous infusion, localized perfusion

bathing target cells directly, via a catheter, in creams, in lipid compositions (e.g., liposomes),

or by other method or any combination of the forgoing as would be known to one of ordinary

skill in the art (see, for example, Remington's Pharmaceutical Sciences (1990), incorporated

herein by reference). In some embodiments, a composition comprising a nucleic acid (e.g.,

administered to the cell, tissue, organ and/or subject is administered using any suitable

method.

[0060] The term "epigenome" or "epigenetics" refers to the modification and structural

changes within a cell that control the expression of nucleic acids (e.g., engineered nucleic

acids) or genomic information in a cell. Changes to the epigenome occur during, and drive

the processes of embryonic development, disease progression, and aging.

[0061] The The term term "epigenetic "epigenetic clock" clock" may may refer refer to to an an age age estimator estimator or or an an innate innate biological biological

process. In some embodiments, rejuvenating or reversing the epigenetic clock refers to

reducing the estimated age of a cell, tissue, organ, or a subject. The epigenetic clock may be

partially or fully reversed or rejuvenated by any of the methods described herein. In some

embodiments, an age estimator is an epigenetic age estimator. For example, an epigenetic

age estimator may be sets of CpG dinucleotides that when used in combination with a

mathematical algorithm may be used to estimate age of a DNA source, including cells,

organs, or tissues. In some embodiments, an age estimator is a DNA methylation-based

(DNAm) age estimator. In some embodiments, a DNAm age estimator is calculated as an

WO wo 2020/069373 PCT/US2019/053545

age correlation using Pearson correlation coefficient r, between DNA methylation-based

(DNam) age (also known as estimated age) and chronological age. In some embodiments, the

DNA methylation-based (DNAm) age estimator is a single-tissue DNA methylation-based

age estimator. In some emodiments, the DNA methylation-based age estimator is a multi-

tissue DNA methylation-based age estimator. In some embodiments, the DNAm age

estimator is DNAm PhenoAge. See, e.g., Horvath and Raj, Nat Rev Genet. 2018

Jun; 19(6):371-384; Levine Jun;19(6):371-384; Levine et et al., al., Aging Aging (Albany (Albany NY). NY). 2018 2018 Apr Apr 18;10(4):573-591; 18;10(4):573-591; and and the the

Examples below.

[0062] "Epigenetic information" as used herein includes covalent modifications to DNA,

such as 5-methylcytosine(5mC), hydroxymethylcytosine (5hmeC), 5-formylcytosine (fC),

and 5-carboxylcytosine (caC), and to certain proteins, such as lysine acetylation, lysine and

arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and

sumoylation of histone proteins, and the 3D architecture of cells, including TADs

(topologically associated domains) and compartments. Epigenetic information is sometimes

referred to as the "analog" information of the cell.

[0063] "Restoring the expression" of at least one gene in Table 5 to youthful levels is

meant to include increasing the expression of a downregulated gene or decreasing the

expression of an upregulated gene that changes during aging.

[0064] As used herein, the term "cell" is meant not only to include an individual cell but

refers also to the particular tissue or organ from which it originates.

[0065] The term "cellular senescence" refers to a cell that has exited the cell cycle,

displays epigenetic markers consistent with senescence, or expressing senescence cell

markers (e.g. senescence-associated beta-galactosidase, or inflammatory cytokines). Cellular

senescence may be partial or complete.

[0066] The term "gene expression" refers to the degree to which certain genes or all genes

in a cell or tissue are transcribed into RNA. In some instances, the RNA is translated by the

cell into a protein. The epigenome dictates gene expression patterns.

[0067] The term "cellular reprogramming" refers to the process of altering the epigenome

of a cell using reprogramming factors (e.g. reversing or preventing epigenetic changes in

cells that are causes of dysfunction, deterioration, cell death, senescence or aging). Cellular

reprogramming may be complete reprogramming, such that a differentiated cell (e.g., somatic

cell) is reprogrammed to a pluripotent stem cell. Cellular reprogramming may be incomplete,

such that a differentiated cell (e.g., somatic cell) retains its cellular identity (e.g., lineage-

specific stem cell). Cellular reprogramming may be incomplete, e.g., a stem cell is not

WO wo 2020/069373 PCT/US2019/053545

created, such that a cell is rejuvenated, or takes on more youthful attributes (e.g. increased

survival, reduced inflammation, or ability to divide). Cellular reprogramming may provide

additional cellular functions, or prevent cellular aging (e.g., transdifferentiation, or transition

into cellular senescence). Cellular reprogramming may induce temporary or permanent gene

expression changes. In some embodiments, incomplete cellular reprogramming is shown by

the lack of Nanog expression. In some embodiments, cellular reprogramming prevents

senescence from occurring.

[0068] The term "rejuvenating a cell" as used herein is meant to include preventing or

reversing the cellular causes of aging without inducing a pluripotent state. A rejuvenated cell

as used herein includes for example a retinal ganglion cell that expresses RBPMS and or

Brn3a.

[0069] A "pluripotent state" as used herein is meant to include a state in which the cell

expresses at least one stem cell marker such as but not limited to Esrrb, Nanog, Lin28, TRA-

1-60/TRA-1-81/TRA-2-54, SSEA1, or SSEA4. Methods of measuring the expression of

stem cell markers on the cell are known in the art and include the methods described herein.

[0070] The term "transdifferentiation" refers to a process in which one cell type is

changed into another cell type without entering a pluripotent state. Transdifferentiation may

also be referred to as lineage reprogramming or lineage conversion. See, e.g., Cieslar-Pobuda Cielar-Pobuda

et al., Biochim Biophys Acta Mol Cell Res. 2017 Jul; ;1864(7):1359-1369, 1864(7): 1359-1369, whichwhich is herein is herein

incorporated by reference in its entirety.

[0071] The terms "condition," "disease," and "disorder" are used interchangeably. Non-

limiting examples of conditions, diseases, and disorders include acute injuries,

neurodegenerative diseases, chronic diseases, proliferative diseases, cardiovascular diseases,

genetic diseases, inflammatory diseases, autoimmunue diseases, neurological diseases,

hematological diseases, painful conditions, psychiatric disorders, metabolic disorders, chronic

diseases, cancers, aging, age-related diseases, and diseases affecting any tissue in a subject.

For example, age-related conditions include, heart failure, stroke, heart disease,

atherosclerosis, neurodegenerative diseases (e.g., Parkinson's disease and Alzheimer's

disease), cognitive decline, memory loss, diabetes, osteoporosis, arthritis, muscle loss,

hearing loss (partial or total), eye-related conditions (e.g., poor eye sight or retinal disease),

glaucoma, a progeroid syndrome (e.g., Hutchinson-Gilford progeria syndrome), and cancer.

In certain embodiments, the disease is a retinal disease (e.g., macular degeneration). In some

embodiments, an age-related condition is senescence. As a non-limiting example, senescence

of glial cells may be a cause of Alzheimer's disease. See e.g., Bussian, et al., Nature. 2018

WO wo 2020/069373 PCT/US2019/053545

Sep 19. In some instances, the condition is nerve damage. In some instances, the condition

is damage in the central nervous system (CNS). In some instances, the nerve damage is

peripheral nerve damage. In some instances, the nerve damage is neurapraxia, axonotmesis,

or neurotmesis.

[0072] In some instances, a condition increases the DNA methylation-based age of a cell,

a tissue, an organ, and/or a subject relative to a control. In some instances, a condition

increases the DNA methylation-based age of a cell, a tissue, an organ, and/or a subject by at

least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at

least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least

300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least

900%, or at least 1,000% relative to a control. In some instances, the control is a cell, a

tissue, an organ, and/or a subject that does not have the condition. In some instances, the

control is the same cell, tissue, organ, and/or subject prior to having the condition. Without

being bound by a particular theory, any of the methods described herein may be useful in

decreasing the DNA methylation-based age of a diseased cell, a diseased tissue, a diseased

organ, and/or a subject who has, is at risk for, or is suspected of having a disease. In some

instances, the disease increases the DNA-methylation-based age of the cell, tissue, organ,

and/or subject. In some instances, the disease is an injury.

[0073] In some instances, the condition is ageing. In some instances, aging is driven by

epigenetic noise. See, e.g., Oberdoerffer and Sinclair. Nat Rev Mol Cell Biol 8, 692-702,

bi:10.1038/nrm2238 doi: 10.1038/nrm2238 (2007); Oberdoerffer et al. Cell 135, 907-918,

doi: 10.1016/j.cell.2008.10.025 (2008). Without being bound by a particular theory,

mammalian cells may retain a faithful copy of epigenetic information from earlier in life,

analogous to Shannon's "observer" system in Information Theory, essentially a back-up copy

of the original signal to allow for its reconstitution at the receiving end if information is lost

or noise is introduced during transmission. See, e.g., Shannon, The Bell System Technical

Journal 27, 379-423 (1948) for a description of the observer system.

[0074] As used herein, an "ocular disease" or "eye disease" is a disease or condition of the

eye. Non-limiting examples of conditions that affect the eye include Ectropion,

Lagophthalmos, Blepharochalasis, Ptosis, Stye, Xanthelasma, Dermatitis, Demodex,

leishmaniasis, loiasis, onchocerciasis, phthiriasis, (herpes simplex), leprosy, molluscum

contagiosum, tuberculosis, yaws, zoster, impetigo, Dacryoadenitis, Epiphora, exophthalmos,

Conjunctivitis, Scleritis, Keratitis, Corneal ulcer / Corneal abrasion, Snow blindness/Arc eye,

Thygeson's superficial punctate keratopathy, Corneal neovascularization, Fuchs' dystrophy,

WO wo 2020/069373 PCT/US2019/053545

Keratoconus, Keratoconjunctivitis sicca, Iritis, iris, Uveitis, Sympathetic ophthalmia,

Cataract, lens, Chorioretinal inflammation, Focal chorioretinal inflammation, chorioretinitis,

choroiditis, retinitis, retinochoroiditis, Disseminated chorioretinal inflammation, exudative

retinopathy, Posterior cyclitis, Pars planitis, chorioretinal inflammations, Harada's disease,

Chorioretinal inflammation, Chorioretinal choroid, inflammation, Chorioretinal choroid, scars, Macula Chorioretinal scars,scars, Maculaposterior scars, pole posterior pole

(postinflammatory) (post-traumatic), Solar retinopathy, Choroidal degeneration, Atrophy,

Sclerosis, angioid streaks, choroidal dystrophy, Choroideremia, choroidal, areolar,

(peripapillary), Gyrate atrophy, choroid, ornithinaemia, Choroidal haemorrhage, Choroidal

detachment, Chorioretinal, Chorioretinal inflammation, infectious and parasitic diseases,

Chorioretinitis, syphilitic, toxoplasma, tuberculosis, chorioretinal, Retinal detachment, retina,

choroid, distorted vision, Retinoschisis, Hypertensive retinopathy, Diabetic retinopathy,

Retinopathy, Retinopathy of prematurity, Age-related macular degeneration, macula, Macular

degeneration, Bull's Eye Maculopathy, Epiretinal membrane, Peripheral retinal degeneration,

Hereditary retinal dystrophy, Retinitis pigmentosa, Retinal haemorrhage, retinal layers,

Central serous retinopathy, Retinal detachment, retinal disorders, Macular edema, macula,

Retinal disorder, Diabetic retinopathy, Glaucoma, optic neuropathy, ocular hypertension,

open-angle glaucoma, angle-closure glaucoma, Normal Tension glaucoma, open-angle

glaucoma, angle-closure glaucoma, Floaters, Leber's hereditary optic neuropathy, Optic disc

drusen, Strabismus, Ophthalmoparesis, eye muscles, Progressive external ophthaloplegia,

Esotropia, Exotropia, Disorders of refraction, accommodation, Hypermetropia, Myopia,

Astigmatism, Anisometropia, Presbyopia, ophthalmoplegia, Amblyopia, Leber's congenital

amaurosis, Scotoma, Anopsia, Color blindness, Achromatopsia / Maskun, cone cells,

Nyctalopia, Blindness, River blindness, Micropthalmia/coloboma, optic nerve, brain, spinal

cord, Red eye, Argyll Robertson pupil, pupils, Keratomycosis, Xerophthalmia, and Aniridia.

In some embodiments, the ocular disease is acute or chronic eye injury.

[0075] In some embodiments, the ocular disease is a scratched cornea.

[0076] In some embodiments, the ocular disease is glaucoma.

[0077] In some embodiments, an ocular disease is a corneal disease (e.g., a disease

affecting the cornea or corneal cells). In some embodiments, an ocular disease is

acanthamoeba keratitis, ectropion, lagoph amblyopia, anisocoria, astigmatism, Bell's Palsy,

blepharitis, blurry vision, burning eyes, cataracts, macular degeneration, age-related macular

degeneration, diabetic eye disease, glaucoma, dry eye, poor vision (e.g., low vision),

astigmatism, blepharitis, cataract, chalazion, conjunctivitis, diabetic retinopathy, dry eye,

glaucoma, keratitis, keratonconus, macular degeneration, ocular hypertension, pinquecula,

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

pterygium, retinitis pigmentosa, or ocular cancer (e.g., retinoblastoma, melanoma of the eye,

lymphoma of the eye, medulloepithelioma, squamous cell cancer of the conjunctiva).

Examples of corneal diseases include, but are not limited to, corneal neovascularization

(NV), corneal dystrophy, corneal inflammation, corneal abrasion, and corneal fibrosis. In

some embodiments, the ocular disease is Keritaconus. In some embodiments, an ocular

disease is macular degeneration. Additional non-limiting examples of eye diseases may be

found in the International Statistical Classification of Diseases and Related Health Problems

(e.g., VII Diseases of the eye and adnexa).

[0078] An ocular disease may affect any part of the eye and/or adnexa. In some

embodiments, the ocular disease is a disorder of the eyelid, lacrimal system and/or orbit. In

some embodiments, the ocular disease is a disorders of conjunctiva. In some embodiments,

the ocular disease is a disorder of sclera, cornea, iris, and/or ciliary body. In some

embodiments, the ocular disease is a disorder of the lens. In some embodiments, the ocular

disease is a disorder of choroid and/or retina. In some embodiments, the ocular disease is

glaucoma. In some embodiments, the ocular disease is a disorder of vitreous body and/or

globe. In some embodiments, the ocular disease is a disorder of optic nerve and/or visual

pathways. In some embodiments, the ocular disease is a disorder of ocular muscles,

binocular movement, accommodation, and/or refraction. In some embodiments, the ocular

disease is a visual disturbance and/or blindness. In some embodiments, the ocular disease is

associated with aging, for example, vision loss associated with aging, decline in visual acuity

associated with aging, and/or decline in retinal function.

[0079] Any suitable method may be used to measure ocular function. Non-limiting

examples include visual acuity tests, pattern electroretinograms, and pathology.

[0080] The The termterm "genetic "genetic disease" disease" refers refers to atodisease a disease caused caused by one by one or more or more abnormalities abnormalities

in the genome of a subject, such as a disease that is present from birth of the subject. Genetic

diseases may be heritable and may be passed down from the parents' genes. A genetic disease

may also be caused by mutations or changes of the DNAs and/or RNAs of the subject. In

such cases, the genetic disease will be heritable if it occurs in the germline. Exemplary

genetic diseases include, but are not limited to, Aarskog-Scott syndrome, Aase syndrome,

achondroplasia, acrodysostosis, addiction, adreno-leukodystrophy, albinism, ablepharon-

macrostomia syndrome, alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency,

Alport's syndrome, Alzheimer's disease, asthma, autoimmune polyglandular syndrome,

androgen insensitivity syndrome, Angelman syndrome, ataxia, ataxia telangiectasia,

atherosclerosis, attention deficit hyperactivity disorder (ADHD), autism, baldness, Batten

WO wo 2020/069373 PCT/US2019/053545

disease, Beckwith-Wiedemann syndrome, Best disease, bipolar disorder, brachydactyl),

breast cancer, Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease,

Crohn's disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome, colon cancer,

congenital adrenal hyperplasia, Cornelia de Lange syndrome, Costello syndrome, Cowden

syndrome, craniofrontonasal dysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob disease,

cystic fibrosis, deafness, depression, diabetes, diastrophic dysplasia, DiGeorge syndrome,

Down's syndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz syndrome, ectodermal

dysplasia Ellis-van Creveld syndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy,

essential tremor, familial hypercholesterolemia, familial Mediterranean fever, fragile X

syndrome, Friedreich's ataxia, Gaucher disease, glaucoma, glucose galactose malabsorption,

glutaricaciduria, gyrate atrophy, Goldberg Shprintzen syndrome (velocardiofacial syndrome),

Gorlin syndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis, hemophilia,

hereditary motor and sensory neuropathy (HMSN), hereditary non polyposis colorectal

cancer (HNPCC), Huntington's disease, immunodeficiency with hyper-IgM, juvenile onset

diabetes, Klinefelter's syndrome, Kabuki syndrome, Leigh's disease, long QT syndrome,

lung cancer, malignant melanoma, manic depression, Marfan syndrome, Menkes syndrome,

miscarriage, mucopolysaccharide disease, multiple endocrine neoplasia, multiple sclerosis,

muscular dystrophy, myotrophic lateral sclerosis, myotonic dystrophy, neurofibromatosis,

Niemann-Pick disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer,

Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Pendred syndrome, peroneal

muscular atrophy, phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome,

primary biliary cirrhosis, prostate cancer, REAR syndrome, Refsum disease, retinitis

pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia, severe

combined immunodeficiency, sickle cell anemia, spina bifida, spinal muscular atrophy,

spinocerebellar atrophy, sudden adult death syndrome, Tangier disease, Tay-Sachs disease,

thrombocytopenia absent radius syndrome, Townes-Brocks syndrome, tuberous sclerosis,

Turner syndrome, Usher syndrome, von Hippel-Lindau syndrome, Waardenburg syndrome,

Weaver syndrome, Werner syndrome, Williams syndrome, Wilson's disease, xeroderma

piginentosum, a progeroid syndrome (e.g., Hutchinson-Gilford progeria syndrome), and

Zellweger syndrome.

[0081] A "proliferative disease" refers to a disease that occurs due to abnormal growth or

extension by the multiplication of cells (Walker, Cambridge Dictionary of Biology;

Cambridge University Press: Cambridge, UK, 1990). A proliferative disease may be

associated with: 1) the pathological proliferation of normally quiescent cells; 2) the

WO wo 2020/069373 PCT/US2019/053545

pathological migration of cells from their normal location (e.g., metastasis of neoplastic

cells); 3) the pathological expression of proteolytic enzymes such as the matrix

metalloproteinases (e.g., collagenases, gelatinases, and elastases); or 4) the pathological

angiogenesis as in proliferative retinopathy and tumor metastasis. Exemplary proliferative

diseases include cancers (i.e., "malignant neoplasms"), benign neoplasms, angiogenesis,

inflammatory diseases, and autoimmune diseases.

[0082] The terms "neoplasm" and "tumor" are used herein interchangeably and refer to an

abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated

with the growth of a normal tissue. A neoplasm or tumor may be "benign" or "malignant,"

depending on the following characteristics: degree of cellular differentiation (including

morphology and functionality), rate of growth, local invasion, and metastasis. A "benign

neoplasm" is generally well differentiated, has characteristically slower growth than a

malignant neoplasm, and remains localized to the site of origin. In addition, a benign

neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites.

Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas,

acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In

some cases, certain "benign" tumors may later give rise to malignant neoplasms, which may

result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and

these tumors are referred to as "pre-malignant neoplasms." An exemplary pre-malignant

neoplasm is a teratoma. In contrast, a "malignant neoplasm" is generally poorly differentiated

(anaplasia) and has characteristically rapid growth accompanied by progressive infiltration,

invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm

generally has the capacity to metastasize to distant sites. The term "metastasis," "metastatic,"

or "metastasize" refers to the spread or migration of cancerous cells from a primary or

original tumor to another organ or tissue and is typically identifiable by the presence of a

"secondary tumor" or "secondary cell mass" of the tissue type of the primary or original

tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is

located. For example, a prostate cancer that has migrated to bone is said to be metastasized

prostate cancer and includes cancerous prostate cancer cells growing in bone tissue.

[0083] The term "cancer" refers to a class of diseases characterized by the development of

abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy

normal body tissues. See, e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams

& Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic

neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g.,

WO wo 2020/069373 PCT/US2019/053545

lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer;

benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer;

breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast,

mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma,

glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus

cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma;

chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal

adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma;

endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma);

endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g.,

adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular

cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder

cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST);

germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral

cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal

cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g.,

leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute

myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia

(CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-

cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell

HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell

lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic

lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma

(MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT)

lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell

lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic

lymphoma (i.e., Waldenström's macroglobulinemia), hairy cell leukemia (HCL),

immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary

central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic

lymphoma/leukemia, lymphoma/leukemia, peripheral peripheral T-cell T-cell lymphoma lymphoma (PTCL) (PTCL) (e.g., (e.g., cutaneous cutaneous T-cell T-cell lymphoma lymphoma

(CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma,

extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous

panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or

more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain

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disease (e.g., alpha chain disease, gamma chain disease, mu chain disease);

hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic

amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma);

liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g.,

bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer

(NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g.,

systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma;

myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis

(ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic

myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL),

hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis

(NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic

neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer);

ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian

adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic

andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile

cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal

tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms;

prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary

gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),

melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft

tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant

peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma);

sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma;

testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g.,

papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid

cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the

vulva).

[0084] The The termterm "inflammatory "inflammatory disease" disease" refers refers to atodisease a disease caused caused by, by, resulting resulting from, from, or or

resulting in inflammation. The term "inflammatory disease" may also refer to a dysregulated

inflammatory reaction that causes an exaggerated response by macrophages, granulocytes,

and/or T-lymphocytes leading to abnormal tissue damage and/or cell death. An inflammatory

disease can be either an acute or chronic inflammatory condition and can result from

infections or non-infectious causes. Inflammatory diseases include, without limitation,

31

WO wo 2020/069373 PCT/US2019/053545

atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus

erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis,

tendonitis, bursitis, psoriasis, cystic fibrosis, arthrosteitis, rheumatoid arthritis, inflammatory

arthritis, Sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis

(scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus,

pemphigoid, diabetes (e.g., Type I), myasthenia gravis, Hashimoto's thyroiditis, Graves'

disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis,

inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia,

inflammatory dermatoses, usual interstitial pneumonitis (UIP), asbestosis, silicosis,

bronchiectasis, berylliosis, talcosis, pneumoconiosis, sarcoidosis, desquamative interstitial

pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular

interstitial pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related

forms of angiitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses,

hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia,

respiratory tract inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis,

immediate hypersensitivity reactions, asthma, hayfever, allergies, acute anaphylaxis,

rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis,

ischemia (ischemic injury), reperfusion injury, allograft rejection, host-versus-graft rejection,

appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis,

chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis,

enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis,

gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis,

orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis,

pneumonitis, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, testitis,

tonsillitis, urethritis, urocystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, angitis,

chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis,

necrotizing fasciitis, and necrotizing enterocolitis. An ocular inflammatory disease includes,

but is not limited to, post-surgical inflammation. In some embodiments, the inflammatory

disease is inflammaging (e.g., inflammation that is a side effect of aging).

[0085] An "autoimmune disease" refers to a disease arising from an inappropriate immune

response of the body of a subject against substances and tissues normally present in the body.

In other words, the immune system mistakes some part of the body as a pathogen and attacks

its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or

involve a particular tissue in different places (e.g., Goodpasture's disease which may affect

WO wo 2020/069373 PCT/US2019/053545

the basement membrane in both the lung and kidney). The treatment of autoimmune diseases

is typically with immunosuppression, e.g., medications which decrease the immune response.

Exemplary autoimmune diseases include, but are not limited to, glomerulonephritis,

Goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, peri-arteritis nodosa,

systemic lupus erythematosis, rheumatoid arthritis, psoriatic arthritis, systemic lupus

erythematosis, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis,

anti-phospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated

vasculitis (e.g., Wegener's granulomatosis, microscopic polyangiitis), uveitis, Sjogren's

syndrome, Crohn's disease, Reiter's syndrome, ankylosing spondylitis, Lyme disease,

Guillain-Barré syndrome, Hashimoto's thyroiditis, and cardiomyopathy.

[0086] The The term term "liverdisease" "liver disease" or or "hepatic "hepaticdisease" disease"refers to damage refers to or to to damage a disease of the of the or a disease

liver. Non-limiting examples of liver disease include intrahepatic cholestasis (e.g., alagille

syndrome, biliary liver cirrhosis), fatty liver (e.g., alcoholic fatty liver, Reye's syndrome),

hepatic vein thrombosis, hepatolenticular degeneration (i.e., Wilson's disease), hepatomegaly,

liver abscess (e.g., amebic liver abscess), liver cirrhosis (e.g., alcoholic, biliary, and

experimental liver cirrhosis), alcoholic liver diseases (e.g., fatty liver, hepatitis, cirrhosis),

parasitic liver disease (e.g., hepatic echinococcosis, fascioliasis, amebic liver abscess),

jaundice (e.g., hemolytic, hepatocellular, cholestatic jaundice), cholestasis, portal

hypertension, liver enlargement, ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis,

chronic hepatitis (e.g., autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced

chronic hepatitis), toxic hepatitis, viral human hepatitis (e.g., hepatitis A, hepatitis B,

hepatitis C, hepatitis D, hepatitis E), granulomatous hepatitis, secondary biliary cirrhosis,

hepatic encephalopathy, varices, primary biliary cirrhosis, primary sclerosing cholangitis,

hepatocellular adenoma, hemangiomas, bile stones, liver failure (e.g., hepatic

encephalopathy, acute liver failure), angiomyolipoma, calcified liver metastases, cystic liver

metastases, fibrolamellar hepatocarcinoma, hepatic adenoma, hepatoma, hepatic cysts (e.g.,

Simple cysts, Polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst),

mesenchymal tumors (mesenchymal hamartoma, infantile hemangioendothelioma,

hemangioma, peliosis hepatis, lipomas, inflammatory pseudotumor), epithelial tumors (e.g.,

bile duct hamartoma, bile duct adenoma), focal nodular hyperplasia, nodular regenerative

hyperplasia, hepatoblastoma, hepatocellular carcinoma, cholangiocarcinoma,

cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma,

hemangioendothelioma, embryonal sarcoma, fibrosarcoma, leiomyosarcoma,

rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary

PCT/US2019/053545

lymphoma, lymphoma, peliosis peliosis hepatis, hepatis, erythrohepatic erythrohepatic porphyria, porphyria, hepatic hepatic porphyria porphyria (e.g., (e.g., acute acute

intermittent porphyria, porphyria cutanea tarda), and Zellweger syndrome.

[0087] The term "spleen disease" refers to a disease of the spleen. Example of spleen

diseases include, but are not limited to, splenomegaly, spleen cancer, asplenia, spleen trauma,

idiopathic purpura, Felty's syndrome, Hodgkin's disease, and immune-mediated destruction

of the spleen.

[0088] The term "lung disease" or "pulmonary disease" refers to a disease of the lung.

Examples of lung diseases include, but are not limited to, bronchiectasis, bronchitis,

bronchopulmonary dysplasia, interstitial lung disease, occupational lung disease, emphysema,

cystic fibrosis, acute respiratory distress syndrome (ARDS), severe acute respiratory

syndrome (SARS), asthma (e.g., intermittent asthma, mild persistent asthma, moderate

persistent asthma, severe persistent asthma), chronic bronchitis, chronic obstructive

pulmonary disease (COPD), emphysema, interstitial lung disease, sarcoidosis, asbestosis,

aspergilloma, aspergillosis, pneumonia (e.g., lobar pneumonia, multilobar pneumonia,

bronchial pneumonia, interstitial pneumonia), pulmonary fibrosis, pulmonary tuberculosis,

rheumatoid lung disease, pulmonary embolism, and lung cancer (e.g., non-small-cell lung

carcinoma (e.g., adenocarcinoma, squamous-cell lung carcinoma, large-cell lung carcinoma),

small-cell lung carcinoma).

[0089] A "hematological disease" includes a disease which affects a hematopoietic cell or

tissue. Hematological diseases include diseases associated with aberrant hematological

content and/or function. Examples of hematological diseases include diseases resulting from

bone marrow irradiation or chemotherapy treatments for cancer, diseases such as pernicious

anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia,

sideroblastic anemia, anemia associated with chronic infections such as malaria,

trypanosomiasis, HTV, hepatitis virus or other viruses, myelophthisic anemias caused by

marrow deficiencies, renal failure resulting from anemia, anemia, polycythemia, infectious

mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), acute myeloid leukemia

(AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL),

polycythemia vera, lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic

leukemia, Wilm's tumor, Ewing's sarcoma, retinoblastoma, hemophilia, disorders associated

with an increased risk of thrombosis, herpes, thalassemia, antibody-mediated disorders such

as transfusion reactions and erythroblastosis, mechanical trauma to red blood cells such as

micro-angiopathic hemolytic anemias, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, infections by parasites such as Plasmodium, chemical injuries from, e.g., lead poisoning, and hypersplenism.

[0090] The term "neurological disease" refers to any disease of the nervous system,

including diseases and injuries that involve the central nervous system (brain, brainstem and

cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic

nervous system (parts of which are located in both central and peripheral nervous system).

Neurodegenerative diseases refer to a type of neurological disease marked by the loss of

nerve cells, including, but not limited to, Alzheimer's disease, Parkinson's disease,

amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), and

Huntington's disease. Examples of neurological diseases include, but are not limited to,

vascular dementias, stroke, headache, stupor and coma, dementia, seizure, sleep disorders,

trauma, infections, neoplasms, neuro-ophthalmology, movement disorders, demyelinating

diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular

junctions. Addiction and mental illnesses include, but are not limited to, bipolar disorder and

schizophrenia, are also included in the definition of neurological diseases. Further examples

of neurological diseases include acquired epileptiform aphasia; acute disseminated

encephalomyelitis; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardi

syndrome; Alexander disease; Alpers' disease; alternating hemiplegia; Alzheimer's disease;

amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia;

aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; arteriovenous

malformation; Asperger syndrome; ataxia telangiectasia; attention deficit hyperactivity

disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet's disease; Bell's

palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial

hypertension; Binswanger's disease; blepharospasm; Bloch Sulzberger syndrome; brachial

plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme);

spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome (CTS);

causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral

aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy;

Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain;

Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy (CIDP);

chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including

persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial

arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative trauma disorders; Cushing's

syndrome; cytomegalic inclusion body disease (CIBD); cytomegalovirus infection; dancing

WO wo 2020/069373 PCT/US2019/053545

eyes-dancing feet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier's

syndrome; Dejerine-Klumpke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse

sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic

encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal

angiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's syndrome;

fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich's ataxia;

frontotemporal dementia and other "tauopathies"; Gaucher's disease; Gerstmann's syndrome;

giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre

syndrome; HTLV-1 associated myelopathy; Hallervorden-Spatz disease; head injury;

headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactica

polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated

dementia and neuropathy (see also neurological manifestations of AIDS); holoprosencephaly;

Huntington's disease and other polyglutamine repeat diseases; hydranencephaly;

hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion

body myositis; incontinentia pigmenti; infantile; phytanic acid storage disease; Infantile

Refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial

hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne

syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg-Welander disease; kuru;

Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateral

medullary (Wallenberg) syndrome; learning disabilities; Leigh's disease; Lennox-Gastaut

syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; lissencephaly;

locked-in syndrome; Lou Gehrig's disease (aka motor neuron disease or amyotrophic lateral

sclerosis); lumbar disc disease; lyme disease-neurological sequelae; Machado-Joseph disease;

macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease;

meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller

Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic

amyotrophy; motor neurone disease; moyamoya disease; mucopolysaccharidoses; multi-

infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating

disorders; multiple system atrophy with postural hypotension; muscular dystrophy;

myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonic encephalopathy of infants;

myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic

malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus;

neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick

disease; O'Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism

WO wo 2020/069373 PCT/US2019/053545

sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic

neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Parkinson's disease;

paramyotonia congenita; paraneoplastic diseases; paroxysmal attacks; Parry Romberg

syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful

neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental

disorders; photic sneeze reflex; phytanic acid storage disease; Pick's disease; pinched nerve;

pituitary tumors; polymyositis; porencephaly; Post-Polio syndrome; postherpetic neuralgia

(PHN); postinfectious encephalomyelitis; postural hypotension; Prader-Willi syndrome;

primary lateral sclerosis; prion diseases; progressive; hemifacial atrophy; progressive

multifocal leukoencephalopathy; progressive sclerosing poliodystrophy; progressive

supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (Type I and Type II);

Rasmussen's Encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease;

repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-

associated myelopathy; Rett syndrome; Reye's syndrome; Saint Vitus Dance; Sandhoff

disease; Schilder's disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome;

shingles; Shy-Drager syndrome; Sjogren's syndrome; sleep apnea; Soto's syndrome;

spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; stiff-

person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis;

subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; sydenham chorea;

syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered

spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; tic douloureux; Todd's

paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform

encephalopathies; transverse myelitis; traumatic brain injury; tremor; trigeminal neuralgia;

tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia);

vasculitis including temporal arteritis; Von Hippel-Lindau Disease (VHL); Wallenberg's

syndrome; Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;

Wilson's disease; and Zellweger syndrome.

[0091] The term "musculoskeletal disease" or "MSD" refers to an injury and/or pain in a

subject's joints, ligaments, muscles, nerves, tendons, and structures that support limbs, neck,

and back. In certain embodiments, an MSD is a degenerative disease. In certain

embodiments, an MSD includes an inflammatory condition. Body parts of a subject that may

be associated with MSDs include upper and lower back, neck, shoulders, and extremities

(arms, legs, feet, and hands). In certain embodiments, an MSD is a bone disease, such as

achondroplasia, acromegaly, bone callus, bone demineralization, bone fracture, bone marrow wo 2020/069373 WO PCT/US2019/053545 disease, bone marrow neoplasm, dyskeratosis congenita, leukemia (e.g., hairy cell leukemia, lymphocytic leukemia, myeloid leukemia, Philadelphia chromosome-positive leukemia, plasma cell leukemia, stem cell leukemia), systemic mastocytosis, myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, myeloid sarcoma, myeloproliferative disorders, multiple myeloma, polycythemia vera, pearson marrow-pancreas syndrome, bone neoplasm, bone marrow neoplasm, Ewing sarcoma, osteochondroma, osteoclastoma, osteosarcoma, brachydactyly, Camurati-Engelmann syndrome, Craniosynostosis, Crouzon craniofacial dysostosis, dwarfism, achondroplasia, bloom syndrome, Cockayne syndrome,

Ellis-van Creveld syndrome, Seckel syndrome, spondyloepiphyseal dysplasia,

spondyloepiphyseal dysplasia congenita, Werner syndrome, hyperostosis, osteophyte,

Klippel-Trenaunay-Weber syndrome, Marfan syndrome, McCune-Albright syndrome,

osteitis, osteoarthritis, osteochondritis, osteochondrodysplasia, Kashin-Beck disease, Leri-

Weill dyschondrosteosis, osteochondrosis, osteodystrophy, osteogenesis imperfecta,

osteolysis, Gorham-Stout syndrome, osteomalacia, osteomyelitis, osteonecrosis, osteopenia,

osteopetrosis, osteoporosis, osteosclerosis, otospondylomegaepiphyseal dysplasia,

pachydermoperiostosis, Paget disease of bone, Polydactyly, Meckel syndrome, rickets,

Rothmund-Thomson syndrome, Sotos syndrome, spondyloepiphyseal dysplasia,

spondyloepiphyseal dysplasia congenita, syndactyly, Apert syndrome, syndactyly type II, or or

Werner syndrome. In certain embodiments, an MSD is a cartilage disease, such as cartilage

neoplasm, osteochondritis, osteochondrodysplasia, Kashin-Beck disease, or Leri-Weill

dyschondrosteosis. In certain embodiments, an MSD is hernia, such as intervertebral disk

hernia. In certain embodiments, an MSD is a joint disease, such as arthralgia, arthritis (e.g.,

gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome), Lyme disease,

osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty

syndrome, synovitis, Blau syndrome, nail-patella syndrome, spondyloarthropathy, reactive

arthritis, Stickler syndrome, synovial membrane disease, synovitis, or Blau syndrome. In

certain embodiments, an MSD is Langer-Giedion syndrome. In certain embodiments, an

MSD is a muscle disease, such as Barth syndrome, mitochondrial encephalomyopathy,

MELAS syndrome, MERRF syndrome, MNGIE syndrome, mitochondrial myopathy,

Kearns-Sayre syndrome, myalgia, fibromyalgia, polymyalgia rheumatica, myoma, myositis,

dermatomyositis, neuromuscular disease, Kearns-Sayre syndrome, muscular dystrophy,

myasthenia, congenital myasthenic syndrome, Lambert-Eaton myasthenic syndrome,

myasthenia gravis, myotonia, myotonia congenita, spinal muscular atrophy, tetany,

ophthalmoplegia, or rhabdomyolysis. In certain embodiments, an MSD is Proteus syndrome.

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

In certain embodiments, an MSD is a rheumatic diseases, such as arthritis (e.g., gout (e.g.,

Kelley-Seegmiller syndrome, Lesch-Nyhan lyme disease)), osteoarthritis, psoriatic arthritis,

reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau

syndrome, gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome), polymyalgia

rheumatica, rheumatic fever, rheumatic heart disease, or Sjogren syndrome. In certain

embodiments, an MSD is Schwartz-Jampel syndrome. In certain embodiments, an MSD is a

skeleton disease, such as Leri-Weill dyschondrosteosis, skeleton malformations, Melnick-

Needles syndrome, pachydermoperiostosis, Rieger syndrome, spinal column disease,

intervertebral disk hernia, scoliosis, spina bifida, spondylitis, ankylosing spondylitis,

spondyloarthropathy, reactive arthritis, spondyloepiphyseal dysplasia, spondyloepiphyseal

dysplasia congenita, or spondylosis. In some embodiments, the disease is a musculoskeletal

disease.

[0092] A "painful condition" includes, but is not limited to, neuropathic pain (e.g.,

peripheral neuropathic pain), central pain, deafferentiation pain, chronic pain (e.g., chronic

nociceptive pain, and other forms of chronic pain such as post-operative pain, e.g., pain

arising after hip, knee, or other replacement surgery), pre-operative pain, stimulus of

nociceptive receptors (nociceptive pain), acute pain (e.g., phantom and transient acute pain),

noninflammatory pain, inflammatory pain, pain associated with cancer, wound pain, burn

pain, pain, postoperative postoperativepain, painpain pain, associated with medical associated procedures, with medical pain resulting procedures, pain from resulting from

pruritus, painful bladder syndrome, pain associated with premenstrual dysphoric disorder

and/or premenstrual syndrome, pain associated with chronic fatigue syndrome, pain

associated with pre-term labor, pain associated with withdrawal symptoms from drug

addiction, joint pain, arthritic pain (e.g., pain associated with crystalline arthritis,

osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis or

Reiter's arthritis), lumbosacral pain, musculo-skeletal pain, headache, migraine, muscle ache,

lower back pain, neck pain, toothache, dental/maxillofacial pain, visceral pain and the like.

One or more of the painful conditions contemplated herein can comprise mixtures of various

types of pain provided above and herein (e.g. nociceptive pain, inflammatory pain,

neuropathic pain, etc.). In some embodiments, a particular pain can dominate. In other

embodiments, the painful condition comprises two or more types of pains without one

dominating. A skilled clinician can determine the dosage to achieve a therapeutically

effective amount for a particular subject based on the painful condition.

[0093] The term "psychiatric disorder" refers to a disease of the mind and includes

diseases and disorders listed in the Diagnostic and Statistical Manual of Mental Disorders -

WO wo 2020/069373 PCT/US2019/053545

Fourth Edition (DSM-IV), published by the American Psychiatric Association, Washington

D.C. (1994). Psychiatric disorders include, but are not limited to, anxiety disorders (e.g.,

acute stress disorder agoraphobia, generalized anxiety disorder, obsessive-compulsive

disorder, panic disorder, posttraumatic stress disorder, separation anxiety disorder, social

phobia, and specific phobia), childhood disorders, (e.g., attention-deficit/hyperactivity

disorder, conduct disorder, and oppositional defiant disorder), eating disorders (e.g., anorexia

nervosa and bulimia nervosa), mood disorders (e.g., depression, bipolar disorder, cyclothymic

disorder, dysthymic disorder, and major depressive disorder), personality disorders (e.g.,

antisocial personality disorder, avoidant personality disorder, borderline personality disorder,

dependent personality disorder, histrionic personality disorder, narcissistic personality

disorder, obsessive-compulsive personality disorder, paranoid personality disorder, schizoid

personality disorder, and schizotypal personality disorder), psychotic disorders (e.g., brief

psychotic disorder, delusional disorder, schizoaffective disorder, schizophreniform disorder,

schizophrenia, and shared psychotic disorder), substance-related disorders (e.g., alcohol

dependence, amphetamine dependence, cannabis dependence, cocaine dependence,

hallucinogen dependence, inhalant dependence, nicotine dependence, opioid dependence,

phencyclidine dependence, and sedative dependence), adjustment disorder, autism, delirium,

dementia, multi-infarct dementia, learning and memory disorders (e.g., amnesia and age-

related memory loss), and Tourette's disorder.

[0094] The term "metabolic disorder" refers to any disorder that involves an alteration in

the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or a combination

thereof. A metabolic disorder is associated with either a deficiency or excess in a metabolic

pathway resulting in an imbalance in metabolism of nucleic acids, proteins, lipids, and/or

carbohydrates. Factors affecting metabolism include, and are not limited to, the endocrine

(hormonal) control system (e.g., the insulin pathway, the enteroendocrine hormones including

GLP-1, PYY or the like), the neural control system (e.g., GLP-1 in the brain), or the like.

Examples of metabolic disorders include, but are not limited to, diabetes (e.g., Type I

diabetes, Type II diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin

resistance, and obesity.

[0095] In some embodiments, a disease is characterized by cellular dysfunction. For

example, a disease may be a mitochondrial disease. Non-limiting mitochondrial diseases

include Freidrich's ataxia, alphers disease, barth syndrome, beta-oxidation defects, carnitine

deficiency, CPT I deficiency, and mitochondrial DNA depletion. Cellular dysfunction may

WO wo 2020/069373 PCT/US2019/053545

include mitochondria dysfunction, RNA replication dysfunction, DNA replication

dysfunction, translation dysfunction, and/or protein folding dysfunction.

[0096] In some In some embodiments, embodiments, the the disease disease or condition or condition by abywood, a wood, bleeding bleeding out,out, injuries injuries

(e.g., broken bones, gunshot wound, cut, scarring during surgery (e.g., cesarean).

[0097] In some embodiments, the disease is an infectious disease (e.g., a disease caused

by a pathogen and/or virus). Non-limiting examples of infectious diseases include

tuberculosis, HIV/AIDS, rabies, plague, cholera, dengue fever, measles, malaria, meningitis,

whooping cough, Lyme disease, influenza, hepatitis C, typhoid fever, and poliomyelitis.

[0098] "Cellular causes of aging" as used herein include loss or modification of epigenetic

information.

[0099] The The terms terms "c-Myc" "c-Myc" or "Myc" or "Myc" refer refer to atonuclear a nuclear phosphoprotein phosphoprotein thatthat has has beenbeen

implicated in cell cycle progression. c-Myc is capable of forming a heterodimer with the

transcription factor MAX and the heterodimer is capable of binding to an E box consequence

sequence on nucleic acids (e.g., engineered nucleic acids) to regulate transcription of target

genes. In certain embodiments, a nucleotide sequence encoding c-Myc comprises a sequence

that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical

to a sequence as described in the NCBI RefSeq database under accession number

NM_001354870.1 or NM_002467.5. In certain embodiments, an amino acid sequence

encoding c-Myc comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%,

90%, 95%, 98%, 99%, or 100%) identical to NP_002458.2 or NP_001341799.1. In certain

embodiments, the methods comprise inducing expression of OCT4; KLF4; SOX2; or any

combination thereof in the absence of inducing c-Myc expression or in the absence of

activating c-Myc. Absence of inducing c-Myc expression may refer to absence of substantial

induction of c-Myc expression over endogenous levels of c-Myc expression in a cell, tissue,

subject, or any combination thereof. Absence of substantial induction of c-Myc expression as

compared to endogenous levels of c-Myc expression in a cell, tissue, subject, or any

combination thereof, may refer to increasing c-Myc expression by less than 70%, less than

60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any

values in between as compared to endogenous levels of c-Myc expression in the cell, tissue,

subject, or any combination thereof. Absence of activating c-Myc expression may refer to

absence of substantial activation of c-Myc (e.g., activity) over endogenous c-Myc activity in

a cell, tissue, subject, or any combination thereof. Absence of substantial induction of c-Myc

activity as compared to endogenous c-Myc activity in a cell, tissue, subject, or any

combination thereof, may refer to increasing c-Myc activity by less than 70%, less than 60%,

WO wo 2020/069373 PCT/US2019/053545

less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or any values in

between as compared to endogenous c-Myc activity in the cell, tissue, subject, or any

combination thereof.

[00100] The terms "effective amount" and "therapeutically effective amount," as used

herein, refer to the amount or concentration of an inventive compound, that, when

administered administered to to aa subject, subject, is is effective effective to to at at least least partially partially treat treat aa condition condition from from which which the the

subject is suffering.

[00101] As used herein, a protein that is "functional" or "active" is one that retains its

biological activity (e.g., capable of acting as a transcription factor or as an inducing agent).

Conversely, a protein that is not functional or is inactive is one that is not capable of

performing one or more of its wild-type functions.

[00102] The The termterm "gene"refers "gene" refers to to aa nucleic nucleic acid acid(e.g., engineered (e.g., nucleic engineered acid) acid) nucleic fragment fragment

that expresses a protein, including regulatory sequences preceding (5' non-coding sequences)

and following 'non-coding sequences) (3' non-coding the the sequences) coding sequence. coding "Native sequence. gene" "Native refers gene" to a refers togene a gene

as found in nature with its own regulatory sequences. "Chimeric gene" or "chimeric

construct" refers to any gene or a construct, not a native gene, comprising regulatory and

coding sequences that are not found together in nature. Accordingly, a chimeric gene or

chimeric construct may comprise regulatory sequences and coding sequences that are derived

from different sources, or regulatory sequences and coding sequences derived from the same

source, but arranged in a manner different than that found in nature. "Endogenous gene"

refers to a native gene in its natural location in the genome of an organism. A "foreign" gene

refers to a gene not normally found in the host organism, but which is introduced into the host

organism by gene transfer. Foreign genes can comprise native genes inserted into a non-

native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the

genome by a transformation procedure.

"Homolog"

[00103] "Homolog" or "homologous" or "homologous" refers refers to sequences to sequences (e.g., (e.g., nucleic nucleic acidacid (e.g., (e.g.,

engineered nucleic acid) or amino acid sequences) that share a certain percent identity (e.g.,

at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%,

at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 71%,

at least 72%, at least73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%,

at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%,

at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,

at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%,

or 100% percent identity). Homologous sequences include but are not limited to paralogous or orthologous sequences. Paralogous sequences arise from duplication of a gene within a genome of a species, while orthologous sequences diverge after a speciation event. A functional homolog retains one or more biological activities of a wild-type protein. In certain embodiments, a functional homolog of OCT4, KLF4, or SOX2 retains at least 10%, at least

20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least

90%, or at least 100% of the biological activity (e.g., transcription factor activity) of a wild-

type counterpart.

[00104] "KLF4"

[00104] "KLF4" may may alsoalso be referred be referred to Kruppel-like to as as Kruppel-like factor factor 4, EZF, 4, EZF, or GKLF or GKLF and and is ais a

zinc-finger transcription factor. KLF4 has been implicated in regulation of differentiation

and proliferation and is capable of interacting with co-activators, including members of the

p300-CBP coactivator family. A KLF4 transcription factor, homolog (e.g., functional

homolog), or variant thereof, as used herein, may be derived from any species, including

humans. In certain embodiments, the nucleic acid (e.g., engineered nucleic acid) encoding

human KLF4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%,

95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., engineered nucleic acid) described

in the NCBI RefSeq database under accession number NM_004235.5 or NM_001314052.1.

Non-limiting examples of KLF4 variants include Krueppel-like factor 4 transcript variant 1

and Krueppel-like factor 4 transcript variant 2. In certain embodiments, KLF4 comprises a

80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 5 or SEQ ID NO: 44.

SEQ ID NO: 5 is a non-limiting example of a nucleotide sequence encoding KLF4 from mus

musculus. SEQ ID NO: 44 is a non-limiting example of a nucleotide sequence encoding

human KLF4. In certain embodiments, KLF4 comprises an amino acid sequence that is at

least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to

NP_001300981.1 or NP_004226.3. In certain embodiments, KLF4 comprises an amino acid

identical to SEQ ID NO: 6. In certain embodiments, KLF4 comprises an amino acid

identical to SEQ ID NO: 45. SEQ ID NO: 6 is a non-limiting example of an amino acid

sequence encoding KLF4 from mus musculus. SEQ ID NO: 45 is a non-limiting example of

an amino acid sequence encoding human KLF4.

"Inverted

[00105] "Inverted terminalrepeats" terminal repeats" or or "ITRs" "ITRs"are arenucleic acid nucleic (e.g., acid engineered (e.g., nucleic engineered nucleic

acid) sequences that are reverse complements of one another. In general, in an AAV vector,

ITRs are found on either side of a cassette (e.g., an expression cassette comprising a nucleic

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

acid (e.g., engineered nucleic acid) encoding OCT4; KLF4; SOX2; or any combination

thereof). In some instances, the cassette encodes an inducing agent. AAV ITRs include ITRs

from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV variants thereof.

[00106] The The terms terms "nucleic "nucleic acid," acid," "polynucleotide", "polynucleotide", "nucleotide "nucleotide sequence", sequence", "nucleic "nucleic acidacid

(e.g., engineered nucleic acid) molecule", "nucleic acid (e.g., engineered nucleic acid)

sequence", and "oligonucleotide" refer to a series of nucleotide bases (also called

"nucleotides") in DNA and RNA, and mean any chain of two or more nucleotides. The terms

"nucleic acid" or "nucleic acid (e.g., engineered nucleic acid) sequence", "nucleic acid (e.g.,

engineered nucleic acid) molecule", "nucleic acid (e.g., engineered nucleic acid) fragment" or

"polynucleotide" may be used interchangeably with "gene", "mRNA encoded by a gene" and

"cDNA".

[00107] The The nucleic nucleic acids(e.g., acids (e.g., engineered engineered nucleic nucleicacids) can can acids) be chimeric mixtures be chimeric or mixtures or

derivatives or modified versions thereof, single-stranded or double-stranded. The

oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for

example, to improve stability of the molecule, its hybridization parameters, etc. A nucleotide

sequence typically carries genetic information, including the information used by cellular

machinery to make proteins and enzymes. These terms include double- or single-stranded

genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and

both sense and antisense polynucleotides. This includes single- and double-stranded

molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic

acids (e.g., engineered nucleic acids) " (PNAs) (PNAs) formed formed byby conjugating conjugating bases bases toto anan amino amino acid acid

backbone. This also includes nucleic acids (e.g., engineered nucleic acids) containing

carbohydrate or lipids. Exemplary DNAs include single-stranded DNA (ssDNA), double-

stranded DNA (dsDNA), plasmid DNA (pDNA), genomic DNA (gDNA), complementary

DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA,

mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen,

repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single-stranded

RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), messenger

RNA (mRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin

RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense

RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA

(ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, viral satellite

RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

(snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid,

ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, and

viral satellite RNA.

[00108] The nucleic acids (e.g., engineered nucleic acids) described herein may be

synthesized by standard methods known in the art, e.g., by use of an automated DNA

synthesizer (such as those that are commercially available from Biosearch, Applied

Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the

method of Stein et al., Nucl. Acids Res., 16, 3209, (1988), methylphosphonate

oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et

al., Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451, (1988)). A number of methods have been

developed for delivering antisense DNA or RNA to cells, e.g., antisense molecules can be

injected directly into the tissue site, or modified antisense molecules, designed to target the

desired cells (antisense linked to peptides or antibodies that specifically bind receptors or

antigens expressed on the target cell surface) can be administered systemically. Alternatively,

RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences

encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a

wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7

or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize

antisense RNA constitutively or inducibly, depending on the promoter used, can be

introduced stably into cell lines. However, it is often difficult to achieve intracellular

concentrations of the antisense sufficient to suppress translation of endogenous mRNAs.

Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense

oligonucleotide is placed under the control of a strong promoter. The use of such a construct

to transfect target cells in the patient will result in the transcription of sufficient amounts of

single stranded RNAs that will form complementary base pairs with the endogenous target

gene transcripts and thereby prevent translation of the target gene mRNA. For example, a

vector can be introduced in vivo such that it is taken up by a cell and directs the transcription

of an antisense RNA. Such a vector can remain episomal or become chromosomally

integrated, as long as it can be transcribed to produce the desired antisense RNA. Such

vectors can be constructed by recombinant DNA technology methods standard in the art.

Vectors can be plasmid, viral, or others known in the art, used for replication and expression

in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any

promoter known in the art to act in mammalian, preferably human, cells. Such promoters can

be inducible or constitutive. Any type of plasmid, cosmid, yeast artificial chromosome, or

WO wo 2020/069373 PCT/US2019/053545

viral vector can be used to prepare the recombinant DNA construct that can be introduced

directly into the tissue site.

[00109] The The nucleic nucleic acids(e.g., acids (e.g., engineered engineered nucleic nucleicacids) may may acids) be flanked by natural be flanked by natural

regulatory (expression control) sequences or may be associated with heterologous sequences,

including promoters, internal ribosome entry sites (IRES) and other ribosome binding site

sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation

sequences, introns, 5'- - and and 3'-non-coding 3'-non-coding regions, regions, and and the the like. like. The The nucleic nucleic acids acids (e.g., (e.g.,

engineered nucleic acids) may also be modified by many means known in the art. Non-

limiting examples of such modifications include methylation, "caps", substitution of one or

more of more ofthe thenaturally occurring naturally nucleotides occurring with anwith nucleotides analog, and internucleotide an analog, and internucleotide

modifications, such as, for example, those with uncharged linkages (e.g., methyl

phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged

linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain

one or more additional covalently linked moieties, such as, for example, proteins (e.g.,

nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine,

psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and

alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl

phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein

may also be modified with a label capable of providing a detectable signal, either directly or

indirectly. Exemplary labels include radioisotopes, fluorescent molecules, epitope tags,

isotopes (e.g., radioactive isotopes), biotin, and the like.

[00110] A "recombinant A "recombinant nucleic nucleic acidacid (e.g., (e.g., engineered engineered nucleic nucleic acid) acid) molecule" molecule" or or

"engineered nucleic acid" is a nucleic acid (e.g., engineered nucleic acid) molecule that has

undergone a molecular biological manipulation, i.e., non-naturally occurring nucleic acid

(e.g., engineered nucleic acid) molecule or genetically engineered nucleic acid (e.g.,

engineered nucleic acid) molecule. Furthermore, the terms "recombinant DNA molecule" or

"engineered nucleic acid" refer to a nucleic acid (e.g., engineered nucleic acid) sequence

which is not naturally occurring, or can be made by the artificial combination of two

otherwise separated segments of nucleic acid (e.g., engineered nucleic acid) sequence, i.e., by

ligating together pieces of DNA that are not normally continuous. By "recombinantly

produced" is meant artificial combination often accomplished by either chemical synthesis

means, or by the artificial manipulation of isolated segments of nucleic acids (e.g.,

engineered nucleic acids), e.g., by genetic engineering techniques using restriction enzymes,

ligases, and similar recombinant techniques as described by, for example, Sambrook et al.,

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; (1989),

or Ausubel et al., Current Protocols in Molecular Biology, Current Protocols (1989), and

DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover) IREL Press,

Oxford, (1985); each of which is incorporated herein by reference.

[00111] SuchSuch manipulation manipulation may may be done be done to replace to replace a codon a codon withwith a redundant a redundant codon codon

encoding the same or a conservative amino acid, while typically introducing or removing a

sequence recognition site. Alternatively, it may be performed to join together nucleic acid

(e.g., engineered nucleic acid) segments of desired functions to generate a single genetic

entity comprising a desired combination of functions not found in nature. Restriction enzyme

recognition sites are often the target of such artificial manipulations, but other site specific

targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, open

reading frames, or other useful features may be incorporated by design.

[00112] "OCT4" may also be referred to as Octamer-binding transcription factor 4, OCT3,

OCT3/4, POU5F1, or POU class 5 homeobox 1 and is a transcription factor that has been

implicated in embryonic development and determination of cell fate. Similar to other OCT

transcription factors, OCT4 is characterized by a bipartite DNA binding domain called a POU

domain. An OCT4 transcription factor, homolog, or variant thereof, as used herein, may be

derived from any species, including humans. In certain embodiments, the nucleic acid (e.g.,

engineered nucleic acid) encoding human OCT4 is at least 70% (e.g., at least 75%, 80%,

85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g., engineered nucleic

acid) described acid) describedinin thethe NCBI RefSeq NCBI underunder RefSeq accession number number accession NM_002701, NM_203289, NM_002701, NM_203289,

NM_001173531, NM_001285986, or NM_001285987. In certain embodiments, the nucleic

acid (e.g., engineered nucleic acid) encoding an OCT4 comprises a sequence that is at least

70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic

acid (e.g., engineered nucleic acid) sequence provided as SEQ ID NO: 1. SEQ ID NO: 1 is a

non-limiting example of a nucleotide sequence encoding OCT4 from mus musculus. In In

certain embodiments, the nucleic acid (e.g., engineered nucleic acid) encoding a human

OCT4 comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%,

98%, 99%, or 100%) identical to a nucleic acid (e.g., engineered nucleic acid) sequence

provided as SEQ ID NO: 40. SEQ ID NO: 40 is a non-limiting example of a nucleotide

sequence encoding human OCT4. Non-limiting examples of OCT4 variants encompassed

herein include POU5F1, transcript variant 1, POU5F1, transcript variant 2, POU5F1,

transcript variant 3, POU5F1, transcript variant 4, and POU5F1 transcript variant 5. In

certain embodiments, the amino acid sequence encoding human OCT4 is at least 70% (e.g., at

WO wo 2020/069373 PCT/US2019/053545

least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a nucleic acid (e.g.,

engineered nucleic acid) described in the NCBI RefSeq under accession number

NP_001167002.1, NP_001272915.1, NP_001272916.1, NP_002692.2, or NP_976034.4. In

certain embodiments, an OCT4 comprises an amino acid sequence that is at least 70% (e.g.,

at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. SEQ

ID NO: 2 is a non-limiting example of an amino acid sequence encoding OCT4 from mus

musculus. In certain embodiments, an OCT4 comprises an amino acid sequence that is at

least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ

ID NO: 41. SEQ ID NO: 41 is a non-limiting example of an amino acid sequence encoding

human OCT4. Other OCT4 transcription factors (e.g., from other species) are known and

nucleic acids (e.g., engineered nucleic acids) encoding OCT4 transcription factors can be

found in publically available databases, including GenBank.

[00113] The term "promoter" refers to a control region of a nucleic acid (e.g., engineered

nucleic acid) sequence at which initiation and rate of transcription of the remainder of a

nucleic acid (e.g., engineered nucleic acid) sequence are controlled. A promoter may also

contain sub-regions at which regulatory proteins and molecules may bind, such as RNA

polymerase and other transcription factors. Promoters may be constitutive, inducible,

activatable, repressible, tissue-specific, or any combination thereof. A promoter drives

expression or drives transcription of the nucleic acid (e.g., engineered nucleic acid) sequence

that it regulates. Herein, a promoter is considered to be "operably linked" when it is in a

correct functional location and orientation in relation to a nucleic acid (e.g., engineered

nucleic acid) sequence it regulates to control ("drive") transcriptional initiation of that

sequence, expression of that sequence, or a combination thereof.

[00114] A promoter may promote ubiquitous expression or tissue-specific expression of

an operably linked nucleic acid (e.g., engineered nucleic acid) sequence from any species,

including humans. In some embodiments, the promoter is a eukaryotic promoter. Non-

limiting examples of eukaryotic promoters include TDH3, PGK1, PKC1, TDH2, PYK1,

TPI1, AT1, CMV, EF1 alpha, SV40, PGK1 (human or mouse), Ubc, human beta actin, CAG,

TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S,

Ubi, H1, and U6, as would be known to one of ordinary skill in the art (see, e.g., Addgene

website: :blog.addgene.org/plasmids-101-the-promoter-region) olog.addgene.org/plasmids-101-the-promoter-region).

Non-limiting

[00115] Non-limiting examples examples of ubiquitous of ubiquitous promoters promoters include include tetracycline-responsive tetracycline-responsive

promoters (under the relevant conditions), CMV (e.g., SEQ ID NO: 48), EF1 alpha, a SV40

promoter, PGK1, Ubc, CAG, human beta actin gene promoter, a RSV promoter (e.g., SEQ ID

WO wo 2020/069373 PCT/US2019/053545

NO: 47), an EFS promoter (e.g., SEQ ID NO: 49), and a promoter comprising an upstream

activating sequence (UAS). In certain embodiments, the promoter is a mammalian promoter.

[00116] Non-limiting examples of tissue-specific promoters include brain-specific, liver-

specific, muscle-specific, nerve cell-specific, lung-specific, heart-specific, bone-specific,

intestine-specific, skin-specific promoters, brain-specific promoters, and eye-specific

promoters. As an example, a muscle-specific promoter is a desmin promoter (e.g., a

identical to SEQ ID NO: 29). Non-limiting examples of eye-specific promoters include

human GRK1 (rhodopsin kinase) promoter (e.g., SEQ ID NO: 50), human CRX (cone rod

homeobox transcription factor) promoter (e.g., SEQ ID NO: 51), and human NRL promoter

(neural retina leucine zipper transcription factor enhancer upstream of the human TK terminal

promoter).

[00117] In some In some embodiments, embodiments, a promoter a promoter is specific is specific for for senescent senescent cells. cells. For For example, example, a a

promoter may specifically induce expression of an operably linked nucleic acid in a senescent

cell and not in non-senescent cells. As a non-limiting example, the p16 promoter may be

used to promote expression of a operably linked nucleic acid in senescent cells.

In some

[00118] In some embodiments,aa promoter embodiments, promoter of of the thepresent presentdisclosure is suitable disclosure for use is suitable in use in for

AAV vectors. See, e.g., U.S. Patent Application Publication No. 2018/0155789, which is

hereby incorporated by reference in its entirety for this purpose.

[00119] Non-limiting examples of constitutive promoters include CP1, CMV, EF1 alpha,

SV40, PGK1, Ubc, human beta actin, beta tubulin, CAG, Ac5, Rosa26 promoter, COLIAL COL1A1

promoter, polyhedrin, TEF1, GDS, CaM3 5S, Ubi, H1, U6, red opsin promoter (red

promoter), rhodopsin promoter (rho promoter), cone arrestin promoter (car promoter),

rhodopsin kinase promoter (rk promoter). An Ubc promoter may comprise a sequence that is

at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ

ID NO: 18. In some instances, the constitutive promoter is a Rosa26 promoter. In some

instances, the constitutive promoter is a COL1A1 promoter. A red opsin promoter may

comprise a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%,

99%, or 100%) identical to SEQ ID NO: 101. A rho promoter may comprise a sequence that

SEQ ID NO: 102. A cone arrestin promoter may comprise a sequence that is at least 70%

(e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 103.

A rhodopsin kinase promoter may comprise a sequence that is at least 70% (e.g., at least

75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 104. A tissue- specific promoter may be used to drive expression of an engineered nucleic acid, including e.g., a nucleic acid encoding a rtTA, tTA, OCT4, KLF4, SOX2, or any combination thereof.

In some embodiments, a tissue-specific promoter is used to drive expression of a rtTA or a

rTA. Ins ome embodiments, a tissue-specific promoter is used to drive expression of OCT4,

KLF4, and SOX2. In some embodiments, the tissue-specific promoter is selected from the

group consisting of SEQ ID NOS: 101-104. In some mebodiments, the hRK promoter is

used to drive expression of OCT4, KLF4, and SOX2.

An "inducible

[00120] An "inducible promoter" is promoter" is one one that that is ischaracterized characterizedby initiating or enhancing by initiating or enhancing

transcriptional activity when in the presence of, influenced by, or contacted by an inducing

agent. An inducing agent may be endogenous or a normally exogenous condition,

compound, agent, or protein that contacts an engineered nucleic acid (e.g., engineered nucleic

acid) in such a way as to be active in inducing transcriptional activity from the inducible

promoter. In certain embodiments, an inducing agent is a tetracycline-sensitive protein (e.g.,

tTA or rtTA, TetR family regulators).

[00121] Inducible Inducible promoters promoters for for use use in accordance in accordance withwith the the present present disclosure disclosure include include any any

inducible promoter described herein or known to one of ordinary skill in the art. Examples of

inducible promoters include, without limitation, chemically/biochemically-regulated and

physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated

promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline

responsive promoter systems, which include a tetracycline repressor protein (TetR, e.g., SEQ

ID NO: 26, or TetRKRAB, e.g., SEQ ID NO: 27), a tetracycline operator sequence (tetO) and

a tetracycline transactivator fusion protein (tTA), and a tetracycline operator sequence (tetO)

and a reverse tetracycline transactivator fusion protein (rtTA)), steroid-regulated promoters

(e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth

ecdysone receptors, and promoters from the steroid/retinoid/thyroid 25 receptor superfamily),

metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind

and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated

promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)),

temperature/heat-inducible promoters (e.g., heat shock promoters), pH-regulated promoters,

and light-regulated promoters. A non-limiting example of an inducible system that uses a

light-regulated promoter is provided in Wang et al., Nat. Methods. 2012 Feb 12;9(3):266-9.

[00122] In certain embodiments, In certain an inducible embodiments, promoter an inducible comprises promoter a tetracycline comprises (Tet)- a tetracycline (Tet)-

responsive element. For example, an inducible promoter may be a TRE3G promoter (e.g., a

WO wo 2020/069373 PCT/US2019/053545

TRE3G promoter that comprises a sequence that is at least 70% (e.g., at least 75%, 80%,

85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 7). As an example, a TRE

(e.g., TRE2) promoter may comprise a nucleic acid (e.g., engineered nucleic acid) sequence

to SEQ ID NO: 23. As an example, a TRE (e.g., P tight) promoter may comprise a nucleic

acid (e.g., engineered nucleic acid) sequence that is at least 70% (e.g., at least 75%, 80%,

85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 24.

[00123] Additional Additional non-limiting non-limiting examples examples of inducible of inducible promoters promoters include include mifepristone- mifepristone-

responsive promoters (e.g., GAL4-Elb GAL4-E1b promoter) and coumermycin-responsive promoters.

See, e.g., Zhao et al., Hum Gene Ther. 2003 Nov 20;14(17):1619-29.

[00124] A "reverse tetracycline transactivator" ("rtTA"), as used herein, is an inducing

agent that binds to a TRE promoter (e.g., a TRE3G, a TRE2 promoter, or a P tight promoter)

in the presence of tetracycline (e.g., doxycycline) and is capable of driving expression of a

transgene that is operably linked to the TRE promoter. rtTAs generally comprise a mutant

tetracycline repressor DNA binding protein (TetR) and a transactivation domain (see, e.g.,

Gossen et al., Science. 1995 Jun 23;268(5218):1766-9 and any of the transactivation domains

listed herein). The mutant TetR domain is capable of binding to a TRE promoter when

bound to tetracycline. See, e.g., U.S. Provisional Application No. 62/738,894, entitled

MUTANT REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, which was filed on September 28, 2018, under attorney docket number

H0824.70300US00, and is herein incorporated by reference in its entirety.

[00125] "SRY-box

[00125] "SRY-box 2" 2" or or "SOX2" "SOX2" is is a member a member of of thethe SRY-related SRY-related HMG-box HMG-box (SOX) (SOX)

family of transcription factors. SOX2 has been implicated in promoting embryonic

development. Members of the SOX (SRY-related HMG-box) family of transcription factors

are characterized by a high mobility group 5 (HMG)-box DNA sequence. This HMG box is

a DNA binding domain that is highly conserved throughout eukaryotic species. A SOX2

transcription transcriptionfactor, homolog factor, or variant homolog thereof, or variant as used as thereof, herein, used may be derived herein, may befrom any derived from any

species, including humans. In certain embodiments, the nucleic acid (e.g., engineered nucleic

acid) encoding SOX2 comprises a sequence that is at least 70% (e.g., at least 75%, 80%,

acid) described in the NCBI RefSeq under accession number NM_011443.4. In certain

embodiments, the nucleic acid (e.g., engineered nucleic acid) encoding a human SOX2

comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%,

99%, or 100%) identical to a nucleic acid (e.g., engineered nucleic acid) described in the

NCBI RefSeq under accession number NM_003106.4. In certain embodiments, SOX2

comprises a nucleic acid (e.g., engineered nucleic acid) sequence that is at least 70% (e.g., at

least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 3 or SEQ ID

NO: 42. SEQ ID NO: 3 is a non-limiting example of a nucleotide sequence encoding SOX2

from mus musculus. SEQ ID NO: 42 is a non-limiting example of a nucleotide sequence

encoding human SOX2. In certain embodiments, the nucleic acid (e.g., engineered nucleic

acid) encoding human SOX2 comprises a sequence that is at least 70% (e.g., at least 75%,

80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to the amino acid sequence described

in the NCBI RefSeq under accession number NP_003097.1. In some instances, SOX2

comprises an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%,

95%, 98%, 99%, or 100%) identical to SEQ ID NO: 4. In some instances, SOX2 comprises

an amino acid sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%,

99%, or 100%) identical to SEQ ID NO: 43. SEQ ID NO: 4 is a non-limiting example of an

amino acid sequence encoding SOX2 from mus musculus. SEQ ID NO: 43 is a non-limiting

example of an amino acid sequence encoding human SOX2.

[00126] A "multicistronic vector" is a vector that encodes more than one amino acid

sequence (e.g., a vector encoding OCT4 and KLF4, OCT4 and SOX2, KLF4 and SOX2, or

OCT4, SOX2, and KL4 (OSK)). A multicistronic vector allows for expression of multiple

amino acid sequences from a nucleic acid (e.g., engineered nucleic acid) sequence. Nucleic

acid (e.g., engineered nucleic acid) sequences encoding each transcription factor (e.g., OCT4,

KLF4, or SOX2) may be connected or separated such that they produce unconnected

proteins. For example, internal ribosome entry sites (IRES) or polypeptide cleavage signals

may be placed between nucleic acid (e.g., engineered nucleic acid) sequences encoding each

transcription factor in a vector. Exemplary polypeptide cleavage signals include 2A peptides

(e.g., T2A, P2A, E2A, and F2A). A 2A peptide may comprise a sequence that is at least 70%

(e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 9.

In some embodiments, an expression vector of the present disclosure is a multicistronic

expression vector.

[00127] "Reversing aging" or "reversing ageing" as used herein refers to modifying the

physical characteristics associated with aging. All animals typically go through a period of

growth and maturation followed by a period of progressive and irreversible physiological

decline ending in death. The length of time from birth to death is known as the life span of an

organism, and each organism has a characteristic average life span. Aging is a physical

WO wo 2020/069373 PCT/US2019/053545

manifestation of the changes underlying the passage of time as measured by percent of

average life span.

[00128] A "subject" to which administration is contemplated includes, but is not limited

to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant,

child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult))

and/or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus

monkeys, rhesus monkeys); commercially relevant mammals, such as cattle, pigs, horses,

sheep, goats, cats, and/or dogs) and birds (e.g., commercially relevant birds, such as chickens,

ducks, geese, and/or turkeys). In certain embodiments, the animal is a mammal. The animal

may be a male or female and at any stage of development. A non-human animal may be a

transgenic animal.

[00129] One One of ordinary of ordinary skillin skill in the the art art would wouldrecognize recognizethat the the that biological age ofage biological a of a

pediatric subject or adult subject may vary depending on the type of animal. As a non-

limiting example, an adult mouse may be 1 year of age, while an adult human may be more

than 21 years of age. In some embodiments, a pediatric subject is less than 21 years of age,

less than 20 years of age, less than 15 years of age, less than 10 years of age, less than 9 years

of age, less than 8 years of age, less than 7 years of age, less than 6 years of age, less than 5

years of age, less than 4 years of age, less than 3 years of age, less than 2 years of age, less

than 1 year of age, less than 10 months of age, less than 9 months of age, less than 8 months

of age, less than 7 months of age, less than 6 months of age, less than 5 months of age, less

than 4 months of age, less than 2 months of age, or less than 1 month of age. In some

embodiments, an adult subject is at least 3 weeks of age, 1 month of age, at least 2 months of

age, at least 3 months of age, at least 4 months of age, at least 5 months of age, at least 6

months of age, at least 7 months of age, at least 8 months of age, at least 9 months of age, at

least 10 months of age, at least 11 months of age, at least 1 year of age, at least 2 years of age, age,

at least 3 years of age, at least 5 years of age, at least 10 years of age, at least 15 years of age,

at least 20 years of age, at least 25 years of age, at least 30 years of age, at least 40 years of

age, at least 50 years of age, at least 55 years of age, at least 60 years of age, at least 65 years

of age, at least 70 years of age, at least 75 years of age, at least 80 years of age, at least 90

years of age, or at least 100 years of age. In some embodiments, a middle-aged adult subject

is between 1 and 6 months of age, between 6 and 12 months of age, between 1 year and 5

years of age, between 5 years and 10 years of age, between 10 and 20 years of age, between

20 and 30 years of age, between 30 and 50 years of age, between 50 and 60 years of age,

between 40 and 60 years of age, between 40 and 50 years of age, or between 45 and 65 years

WO wo 2020/069373 PCT/US2019/053545

of age. In some embodiments, a senior adult subject is at least 1 month of age, at least 2

months of age, at least 3 months of age, at least 4 months of age, at least 5 months of age, at

least 6 months of age, at least 7 months of age, at least 8 months of age, at least 9 months of

age, at least 10 months of age, at least 11 months of age, at least 1 year of age, at least 2 years

of age, at least 3 years of age, at least 5 years of age, at least 10 years of age, at least 15 years

of age, at least 20 years of age, at least 25 years of age, at least 30 years of age, at least 40

years of age, at least 50 years of age, at least 55 years of age, at least 60 years of age, at least

65 years of age, at least 70 years of age, at least 75 years of age, at least 80 years of age, at

least 90 years of age, or at least 100 years of age.

A "terminator"

[00130] A "terminator" oror"terminator "terminator sequence," sequence,"asasused herein, used is ais herein, nucleic acid (e.g., a nucleic acid (e.g.,

engineered nucleic acid) sequence that causes transcription to stop. A terminator may be

unidirectional or bidirectional. It is comprised of a DNA sequence involved in specific

termination of an RNA transcript by an RNA polymerase. A terminator sequence prevents

transcriptional activation of downstream nucleic acid (e.g., engineered nucleic acid)

sequences by upstream promoters. Thus, in certain embodiments, a terminator that ends the

production of an RNA transcript is contemplated.

[00131] The The mostmost commonly commonly usedused typetype of terminator of terminator is aisforward a forward terminator. terminator. WhenWhen

placed downstream of a nucleic acid (e.g., engineered nucleic acid) sequence that is usually

transcribed, a forward transcriptional terminator will cause transcription to abort. In some

embodiments, bidirectional transcriptional terminators may be used, which usually cause

transcription to terminate on both the forward and reverse strand. In some embodiments,

reverse transcriptional terminators may be used, which usually terminate transcription on the

reverse strand only.

[00132] Non-limiting Non-limiting examples examples of mammalian of mammalian terminator terminator sequences sequences include include bovine bovine

growth hormone terminator, and viral termination sequences such as, for example, the SV40

terminator, spy, yejM, secG-leuU, thrLABC, rrnB T1, hisLGDCBHAFI, metZWV, rrnC,

xapR, aspA, and arcA terminator. In certain embodiments, the terminator sequence is SV40

and comprises a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%,

99%, or 100%) identical to SEQ ID NO: 8.

A "Tet-Off"

[00133] A "Tet-Off" system,as system, asused used herein, herein, is isa atype of of type inducible system inducible that is system capable that is capable

of repressing expression of a particular transgene in the presence of tetracycline (e.g.,

doxycycline (DOX)). Conversely, a Tet-Off system is capable of inducing expression of a

particular transgene in the absence of tetracycline (e.g., doxycycline, DOX). In certain

embodiments, a Tet-Off system comprises a tetracycline-responsive promoter operably linked

WO wo 2020/069373 PCT/US2019/053545

to a transgene (e.g., encoding OCT4; KLF4; SOX2; or any combination thereof) and a

tetracycline-controlled transactivator (tTA). The transgene with the tetracycline-responsive

promoter (e.g., TRE3G, P tight, or TRE2) and the tetracycline-controlled transactivator may

be encoded on the same vector or be encoded on separate vectors. See, e.g., U.S. Provisional

Application No. 62/738,894, entitled MUTANT REVERSE TETRACYCLINE

TRANSACTIVATORS FOR EXPRESSION OF GENES, which was filed on September 28, 2018, under attorney docket number H0824.70300US00, and is herein incorporated by

reference in its entirety.

[00134] A "Tet-On" system, as used herein, is a type of inducible system that is capable of

inducing expression of a particular transgene in the presence of tetracycline (e.g.,

doxycycline (DOX)). In certain embodiments, a Tet-On system comprises a tetracycline-

responsive promoter operably linked to a transgene (e.g., encoding OCT4; KLF4; SOX2; or

any combination thereof) and a reverse tetracycline-controlled transactivator (rtTA). For

example, the rtTA may be rtTA3, rtTA4, or variants thereof. In certain embodiments, a

nucleic acid (e.g., engineered nucleic acid) encoding rtTA3 comprises a sequence that is at

least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least

100%) identical to SEQ ID NO: 10. In certain embodiments, an amino acid sequence

encoding rtTA3 comprises a sequence that is at least 70% (e.g., at least 75%, at least 80%, at

least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100%) identical to

(SEQ ID NO: 11). In certain embodiments, a nucleic acid (e.g., engineered nucleic acid)

encoding rtTA4 comprises a sequence that is at least 70% (e.g., at least 75%, at least 80%, at

SEQ ID NO: 12. In certain embodiments, an amino acid sequence encoding rtTA4 comprises

a sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at

least 95%, at least 98%, at least 99%, or at least 100%) identical to (SEQ ID NO: 13). The

expression cassette encoding a tetracycline-responsive promoter (e.g., a promoter comprising

a TRE, including TRE3G, P tight, and TRE2) and a reverse tetracycline-controlled

transactivator may be encoded on the same vector or be encoded on separate vectors. See,

e.g., U.S. Provisional Application No. 62/738,894, entitled MUTANT REVERSE

TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, which was filed on September 28, 2018, under attorney docket number H0824.70300US00, and is herein

incorporated by reference in its entirety.

[00135] The term "tissue" refers to any biological tissue of a subject (including a group of

cells, a body part, or an organ) or a part thereof, including blood and/or lymph vessels, which

WO wo 2020/069373 PCT/US2019/053545

is the object to which a compound, particle, and/or composition of the invention is delivered.

A tissue may be an abnormal, damaged, or unhealthy tissue, which may need to be treated. A

tissue may also be a normal or healthy tissue that is under a higher than normal risk of

becoming abnormal or unhealthy, which may need to be prevented. In certain embodiments,

the tissue is considered healthy but suboptimal for performance or survival in current or

future conditions. For example, in agricultural practice, environmental conditions including

weather and growing conditions (e.g., nutrition) may benefit from any of the methods

described herein. In certain embodiments, the tissue is the central nervous system. In certain

embodiments, the tissue refers to tissue from the In certain embodiments, the cell or tissue is

from eye, ear, nose, mouth including gum and roots of teeth, bone, lung, breast, udder,

pancreas, stomach, oesophagus, muscle including cardiac muscle, liver, blood vessel, skin

including hair, heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or

intestine. In certain embodiments, the tissue is damaged (e.g., due to a congenital defect, an

injury, an accident, or an iatrogenic injury) and/or is aged tissue. In certain embodiments, the

tissue is a deep tissue that is reachable with a fiber optic probe.

[00136] The term "tetracycline repressor" or "TetR" refers to a protein that is capable of

binding to a Tet-O sequence (e.g., a Tet-O sequence in a TRE, e.g., a Tet-O sequence may

comprise SEQ ID NO: 19) in the absence of tetracycline (e.g., doxycycline) and prevents

binding of rtTA (e.g., rtTA3, rtTA4, or variants thereof) in the absence of tetracycline (e.g.,

doxycycline). TetRs prevent gene expression from promoters comprising a TRE in the

absence of tetracycline (e.g., doxycycline). In the presence of tetracycline, TetRs cannot bind

promoters comprising a TRE, and TetR cannot prevent transcription. Non-limiting examples

of TetRs include tetR (e.g., SEQ ID NO: 26), tetRKRAB (e.g., SEQ ID NO: 28). In some

embodiments, a TetR is a TetR fusion (e.g., TRSID, which may be created by fusing TetR to

a mSIN30interacting domain (SID) of Mad1). See, e.g., Zhang et al., J Biol Chem. 2001 Nov

30;276(48):45168-74.

[00137] As used As used herein, herein, a "TRE a "TRE promoter" promoter" is aispromoter a promoter comprising comprising a tetracycline- a tetracycline-

responsive element (TRE). As used herein, a TRE comprises at least one (e.g., at least 2, 3,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) Tet-O sequences. A non-limiting

example of a Tet-O sequence is sequence that is at least 70% (e.g., 75%, 80%, 85%, 90%,

95%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, a TRE

promoter further comprises a minimal promoter located downstream of a tet-O sequence. A

minimal promoter is a promoter that comprises the minimal elements of a promoter (e.g.,

TATA box and transcription initiation site), but is inactive in the absence of an upstream

WO wo 2020/069373 PCT/US2019/053545

enhancer (e.g., sequences comprising Tet-O). As an example, a minimal promoter may be a

minimal CMV promoter that comprises a sequence that is at least 70% (e.g., at least 75%,

80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 20. For example, a

TRE promoter may be a TRE3G promoter (e.g., a TRE3G promoter that comprises a

identical to SEQ ID NO: 7. In some embodiments, a TRE promoter is a TRE2 promoter

comprising a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%,

99%, or 100%) identical to SEQ ID NO: 23. In some embodiments, a TRE promoter is a P

tight promoter comprising a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%,

95%, 98%, 99%, or 100%) identical to SEQ ID NO: 24.

[00138] The The termterm "tissuerepair" "tissue repair" in in the the context contextofofdamaged tissue damaged refers tissue to restoration refers of to restoration of

tissue architecture, function following tissue damage, or a combination thereof. Tissue repair

includes tissue regeneration, cell growth, tissue replacement, and/or rewiring of existing

tissue (reprogramming).

[00139] The The termterm "tissueregeneration" "tissue regeneration" refers referstotoproduction of new production tissue of new or cells tissue within within or cells a a

tissue that are the same type as the tissue of interest (e.g., same type as the damaged tissue or or

cell). In some embodiments, the methods provided herein promote organ regeneration.

[00140] The term "tissue replacement" refers to production of a different type of tissue

compared to the tissue of interest (e.g., connective tissue to replace damaged tissue).

As used

[00141] As used herein, herein, theterms the terms "treatment," "treatment," "treat," "treat,"andand "treating" referrefer "treating" to reversing, to reversing,

alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or

more symptoms thereof, as described herein. In certain embodiments, treatment may be

administered after one or more symptoms have developed. In other embodiments, treatment

may be administered in the absence of symptoms. For example, treatment may be

administered to a susceptible individual prior to the onset of symptoms or may be treated

with another damaging agent (e.g., in light of a history of symptoms, in light of genetic or

other susceptibility factors, a disease therapy, or any combination thereof). Treatment may

also be continued after symptoms have resolved, for example, to prevent or delay their

recurrence.

[00142] The term "variant" refers to a sequence that comprises a modification relative to a

wild-type sequence. Non-limiting modifications in an amino acid sequence include

insertions, deletions, and point mutations. Non-limiting modifications to nucleic acid (e.g.,

engineered nucleic acid) sequences include frameshift mutations, nucleotide insertions, and

nucleotide deletions.

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

[00143] The term "WPRE" refers to a Woodchuck Hepatitis Virus (WHP)

Posttranscriptional Regulatory Element (WPRE). WPREs create tertiary structures in nucleic

acids (e.g., expression vectors) and are capable of enhancing transgene expression (e.g., from

a viral vector). In certain embodiments, a WPRE sequence is at least 70% (e.g., at least 75%,

at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least

100%) identical to SEQ ID NO: 21.

[00144] These and other exemplary substituents are described in more detail in the

Detailed Description, Examples, and claims. The invention is not intended to be limited in

any manner by the above exemplary listing of substituents.

BRIEF DESCRIPTION OF THE DRAWINGS

[00145] FIG. 1 is a schematic with a linear representation of an expression vector

encoding OCT4, SOX2, and KLF4. TRE3G is shown as an exemplary inducible promoter,

and SV40 is shown as an exemplary terminator sequence.

[00146] FIG. 2 is a vector map of TRE3G-OSK-SV40pA, an AAV vector encoding OSK.

Features including the location of sequences encoding OCT4, SOX2, and KLF4 and inverted

terminal repeat sequences (ITRs) are indicated.

[00147] FIG. 3 is a vector map showing the location of restriction enzyme digestion sites

in TRE3G-OSK-SV40pA.

[00148] FIGS. 4A-4AL include a series of schematics mapping the features shown in

FIG. 2 and 3 onto the nucleic acid (e.g., engineered nucleic acid) sequence of TRE3G-OSK-

SV40pA.

[00149] FIGS. 5A-5D show the nucleotide positions and lengths of the nucleic acid (e.g.,

engineered nucleic acid) sequences of the features shown in FIGS. 4A-4AL.

[00150] FIGS. 6A-6C include western blot data showing that different serotypes of AAVs

encoding OSK (TRE3G-OSK-SV40pA, SEQ ID NO: 16) were successfully used in a

doxycycline (DOX)-inducible system to control OSK expression in 293T cells. OCT4,

KLF4, and H3 expression were detected with antibodies. H3 refers to histone 3 and is a

loading control. FIG. 6A shows the effect of doxycycline on protein expression in cells

infected with AAV9 virus harboring the TRE3G-OSK-SV40pA vector and with AAV9 virus

harboring a vector that encodes rtTA3 (tetracycline (Tet)-on system). FIG. 6B shows the

effect of DOX on protein expression in cells infected with AAV2 virus harboring the

TRE3G-OSK-SV40pA TRE3G-OSK-SV40pA vector vector and and with with AAV2 AAV2 virus virus harboring harboring aa vector vector that that encodes encodes tTA tTA

(Tet-Off system). FIG. 6C shows the effect of DOX treatment and DOX removal on protein

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

expression in cells infected with AAV.PHP.b virus harboring the TRE3G-OSK-SV40pA

vector and with AAV.PHP.b virus harboring a vector that encodes rtTA3 (Tet-On system).

The length of DOX treatment (+DOX) or DOX removal (-DOX) in days is indicated in

parenthesis.

[00151] FIGS. 7A-7F include data showing that AAV encoding OSK induced partial

reprogramming and promoted regeneration of optic nerves after nerve crush in an inducible

manner. FIG. 7A includes a series of photos showing that injection of TRE-OSK-SV40 AAV

virus and CAG-tTA AAV virus into mouse retina resulted in expression of KLF4 in mouse

retina ganglion cells (RGCs). RBPMS (RGC marker) and KLF4 staining of an optical

coherence tomography (OCT) section from mouse retina is shown. FIG. 7B includes a series

of photos showing that injection of TRE-OSK-SV40 AAV virus and CAG-tTA AAV virus

resulted in inducible expression of KLF4 and OCT4 in mouse retina. OCT4 and KLF4

staining of a whole retina mount in the absence of doxycycline treatment (top two photos)

and after four days of DOX treatment (bottom two photos) is shown. FIG. 7C shows an

experimental timeline to determine the effect of TRE-OSK-SV40 AAV virus alone or in

combination with CAG-tTA AAV virus on optical nerve regeneration following optic nerve

crush damage. CTB stands for cholera toxin B-subunit ß-subunit and allows for fluorescence imaging

of axons. FIG. 7D shows the co-localization staining of OCT4 and KLF4 from a whole

mount mount retina retinawith TRE-OSK-SV40 with AAV AAV TRE-OSK-SV40 virusvirus injected in combination injected with CAG-tTA, in combination with CAG-tTA,

RBPMS stains retina ganglion cells specifically. FIG. 7E shows fluorescence imaging of

CTB-labeled axons in an optical nerve after crush damage in mouse retina injected with TRE-

OSK-SV40 AAV virus alone (left) or TRE-OSK-SV40 AAV in combination with CAG-tA CAG-tTA

AAV (right). Stars represent the site of the lesion. FIG. 7F shows additional fluorescence

images of optical nerves treated as in FIG. 7E with viruses as indicated.

[00152] FIGS.

[00152] FIGS. 8A-8G 8A-8G shows shows administration administration of of virus virus encoding encoding OSKOSK improved improved RGCRGC axon axon

regeneration after nerve crush injury. FIG. 8A shows the effect of virus encoding tTA in

combination with virus encoding TRE-OSK-SV40 in the absence of DOX (circles, n=9), with

virus encoding TRE-OSK-SV40 in the presence of dox (triangles, n=5), or with virus

encoding d2EGFP (squares, control, n=5) on RGC axon regeneration. The number of

estimated axons per nerve is shown as a function of the distance from the site of injury (um). (µm).

FIG. 8B is an experimental timeline to determine the effect of d2EGFP expression on RGC

axon regeneration. FIG. 8C is a series of images showing CTB-labeled axons from the

experiment outlined in FIG. 8B. FIG. 8D is an experimental timeline to determine the effect

of uninduced OSK expression on axon regeneration. FIG. 8E is a series of images showing

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CTB-labeled axons from the experiment outlined in Fig. 4D. FIG. 8F is an experimental

timeline to determine the effect of induced OSK expression on axon regeneration. FIG. 8G is

a series of images showing CTB-labeled axons from the experiment outlined in FIG. 8F.

Stars indicate the site of the lesion.

[00153] FIGS. 9A-9D show OSK-infected RGCs have a higher survival rate compared to

cells not infected with OSK virus following nerve crush. FIG. 9A shows staining for

RBPMS and GFP in GFP AAV-infected uncrushed RGCs (upper left) and in crushed RGCs

(upper right). staining for RBPMS and KLF4 in OSK AAV-infected uncrushed RGCs (lower

left) and in crushed RGCs (lower right). FIG. 9B shows the ratio of RBPMS (RNA binding

protein with multiple splicing)-positive (+) cells for uncrushed and crushed RGCs infected

with a destabilized form of GFP (d2EGFP) virus or OSK virus. GFP infected RGCs has the

same survival rate as uninfected RGCs, therefore GFP+ RBPMs+% remains the same after

crush injury. OSK infected RGCs had triple the survival rate compared to uninfected RGC,

therefore, KLF4+ RBPMS+% increased after crush injury. FIG. 9C shows survived RGCs

under uncrushed (left) and crushed (right) condition, with OSK virus infection. FIG. 9D

shows the survival of RGCs (RBPMS+) under uncrushed and crushed condition, when they

were infected with d2EGFP virus or OSK virus.

[00154] FIGS. 10A-10B show that OSK-mediated regeneration and protection is

independent of mTOR activation. FIG. 10A is a series of images showing RBPMS and pS6

staining of control and OSK-infected RGCs that were uncrushed or crushed. FIG. 10B is a

graph quantifying the percentage of pS6 positive cells from series of pictures like FIG. 10A.

[00155] FIGS. 11A-11D show that an AAV Tet-On system comprising a CMV-rtTA

vector (SEQ ID NO: 31) induces faster gene expression compared to an AAV Tet-Off system

in retinal cells after nerve crush. FIG. 11A shows an experimental timeline to test the effect

of doxycycline removal on GFP expression in an AAV Tet-Off system. Lines indicate the

length of DOX treatment. Treatments A-D as indicated correspond to photographs 1-4 of

FIG. 7B, respectively. FIG. 11B is a series of photos showing results of the experiment

outlined in FIG. 11A. GFP-positive cells from mouse retina that was infected with virus

encoding tTA and virus encoding TRE-d2EGFP at indicated days of DOX removal are

shown. FIG. 11C shows an experimental timeline to test the effect of doxycycline treatment

on GFP expression in an AAV Tet-On system comprising a CMV-rtTA vector (SEQ ID NO:

31). Lines indicate the length of DOX treatment. Treatments A-C as indicated correspond to

photographs 1-3 of FIG. 11D, respectively. FIG. 11D is a series of photos showing results of

the experiment outlined in FIG. 11C. GFP-positive cells from mouse retina that was infected

PCT/US2019/053545

with virus encoding rtTA and virus encoding TRE-d2EGFP at the indicated days of DOX

treatment are shown.

[00156] FIG. 12 is a vector map showing features in an adeno-associated virus (AAV)

vector encoding reverse tetracycline-transactivator 4 (rtTA4). Ubc is a constitutive promoter

that is operably linked to the nucleic acid (e.g., engineered nucleic acid) encoding rtTA4.

SV40pA is an SV40-derived terminator sequence. The sequence of this vector is provided in

SEQ ID NO: 17.

[00157] FIGS. 13A-13C include data showing that a Tet-On system comprising rtTA4

(SEQ ID NO: 13) has low leakiness in the liver of mice. FIG. 13A is a series of

immunofluorescence images showing expression of KLF4 in the livers of mice that have

been administered AAVs harboring nucleic acids (e.g., engineered nucleic acids) shown in

FIG. FIG. 13B 13B in in the the absence absence of of doxycycline doxycycline (no (no DOX) DOX) and and in in the the presence presence of of doxycycline doxycycline (with (with

DOX). DAPI is a nuclear stain that was used to visualize cells. FIG. 13B is a schematic

depicting the two nucleic acids (e.g., engineered nucleic acids) that were administered to

mice in AAV9 viruses. FIG. 13C is a western blot of liver samples from mice that received

the constructs depicted in FIG. 13B and were treated with no doxycycline or with

doxycycline. OCT4, KLF4, and SOX2 levels were detected as indicated using antibodies.

Actin is shown as a loading control.

[00158] FIG. 14 is a graph comparing the body weights of mice under various treatments

as indicated. WT indicates wild-type mice without exogenous OSK expression. All dead

indicates that OSK transgenic mice treated with doxycycline were all dead.

[00159] FIGS.

[00159] FIGS. 15A-15B include 15A-15B include data data showing showingthat induction that of OCT4, induction KLF4,KLF4, of OCT4, and SOX2 and SOX2

expression reversed aging of mice ear fibroblasts as indicated by expression of histone and

Chaf (Chromatin assembly factor) genes but did not induce Nanog expression. The asterisk

(*) indicates endogenous KLF4 expression from the 293T cell line.

[00160] FIG.FIG. 16 is 16 aiswestern blotblot a western showing thatthat showing an AAV vector an AAV comprising vector a nucleic comprising acidacid a nucleic

(e.g., engineered nucleic acid) sequence that is greater than 4.7 kb between the two ITRs in

the vector has low viral titer when incorporated into an AAV and produces non-functional

AAV. The TRE2-OSK vector is provided as SEQ ID NO: 33. Expression of OCT4, KLF4

and H3 was detected using antibodies. H3 is shown as a loading control. Asterisk (*)

indicates endogenous Klf4 from 293T cell line.

[00161] FIG.FIG. 17 is 17 aiswestern blotblot a western showing thatthat showing administration of modified administration mRNAmRNA of modified

encoding OCT4, SOX2, and KLF4 induced expression of KLF4 and OCT4 in mouse cells.

Antibodies against KLF4, OCT4, GAPDH, and H3 were used to detect indicated proteins.

WO wo 2020/069373 PCT/US2019/053545

[00162] FIG. 18 is a vector map of pAAV2_CMV_rtTA(VP16) (SEQ ID NO: 31). This

vector is a non-limiting example of a vector encoding rtTA.

[00163] FIG. 19 is a vector map of pAAV-MCS-ITA2 pAAV-MCS-tTA2 (or CAG-tA) CAG-tTA)(SEQ (SEQID IDNO: NO:32). 32).

This vector is a non-limiting example of a vector encoding tTA under a CAG promoter.

[00164] FIG.

[00164] FIG. 20 20 is is a vector a vector mapmap of of p-AAV-TetO-OSK-WPRE3-SV50LpA p-AAV-TetO-OSK-WPRE3-SV50LpA (TRE2-OSK, (TRE2-OSK,

pAAV-TRE2-OSK-SV40LpA, pAAV-TRE2-OSK-SV40LpA, or or TRE2-OSK) TRE2-OSK) (SEQ (SEQ ID ID NO: NO: 33). 33). This This vector vector is is aa non- non-

limiting example of an AAV vector comprising a nucleic acid (e.g., engineered nucleic acid)

sequence that is greater than 4.7 kb between the two ITRs in the vector.

[00165] FIG.FIG. 21 is 21 aisseries of images a series showing of images successful showing chemical successful reprogramming chemical of of reprogramming

mouse embryonic fibroblasts.

[00166] FIG. 22 includes a schematic showing a non-limiting example of a Tet-Off

system to express OCT4, SOX2, and KLF4 in the absence of tetracycline (top panel) and a

schematic showing a non-limiting example of a Tet-ON system to express OCT4, SOX2, and

KLF4 (OSK) in the presence of tetracycline (bottom panel).

[00167] FIGS. 23A-23C include data showing that administration of AAV2 virus

encoding OCT4, SOX2, and KLF4 improved axon regeneration and RGC survival in adult

and aged mice two weeks after optic nerve crush. FIG. 23A is a series of images showing

CTB-labeled axons from mice at indicated ages (in months) and comparing the effect

ofAAV2 virus encoding TRE-OSK-SV40 with the effect of AAV2 virus encoding GFP.

Experiments were conducted in the absence of DOX using the Tet-Off system depicted in

FIG. 22, top panel. FIG. 23B quantifies the number of estimated axons per nerve for mice

with the indicated ages and treatments as a function of the distance from the site of injury

(um). (µm). FIG. 23C is a chart showing that OSK increased the survival of RGCs after optic

nerve injury in adult (3 month old) and aged (12 month old) mice compared to control GFP.

The survival of RGCs (RBPMS+) is shown for mice of the indicated ages receiving virus

encoding d2EGFP or OSK.

[00168] FIGS. 24A-24B FIGS. include 24A-24B datadata include showing thatthat showing increasing the the increasing timetime of reprogramming of reprogramming

from two weeks to five weeks improved regeneration in aged mice. FIG. 24A is a series of

photos showing CTB-labeled axons from 12 month old mice five weeks after optic nerve

crush injury. Mice were administered virus encoding GFP or encoding TRE-SV40-OSK and

virus encoding tTA prior to nerve crush injury. FIG. 24B is a graph quantifying the number

of estimated axons per nerve as a function of the distance from the site of injury (um) (µm) from

FIG. 24A.

PCT/US2019/053545

[00169] FIGS. 25A-25C include data showing that induction of OSK expression using

Tet-On and Tet-Off systems even after optic nerve crush injury improved regeneration and

RGC cell survival in mice. FIG. 25A includes schematics showing treatment timelines to

determine the effect of OSK expression before or after optic nerve crush. In the Tet-On

system, induction of OSK expression prior to optic nerve crush injury (pre-injury induction)

and and induction inductionof of OSKOSK expression afterafter expression optic optic nerve crush nerveinjury crush (post-injury induction) induction) injury (post-injury are are

shown (top panel). Doxycycline treatment was used to induce OSK expression. In the Tet-

Off system, suppression of OSK induction with doxycycline treatment prior to optic nerve

crush (pre-injury suppression) and suppression of OSK induction with doxycycline treatment

after optic nerve crush (post-injury suppression) are shown (bottom panel). The shaded lines

on the timeline indicate the length of doxycycline (DOX) treatment. Cholera toxin B-subunit ß-subunit

(CTB) injection for imaging of axons is also shown. FIG. 25B is a chart quantifying the

number of estimated axons per nerve as a function of the distance from the site of injury (um) (µm)

for four-week old (young) mice with no OSK induction (n=4), OSK induction pre-injury only

(n=5), OSK expression suppressed from injury (n=5), and OSK induction post injury (n=5).

The protocols for pre-injury and post-injury induction used were as shown in FIG. 25A. FIG.

25C is a chart quantifying the number of RBPMS+ cells from four-week old (young) mice

with no OSK induction, OSK induction pre-injury only, OSK suppressed from injury, and

OSK induction post injury.

FIGS.

[00170] FIGS. 26A-26E 26A-26E include include datadata showing showing thatthat expression expression of OSK of OSK fromfrom a single a single

transcript improved axon regeneration and retina ganglion cell (RGC) survival two weeks

after optic nerve crush injury compared to expression of OCT4, SOX2, and KLF4 from

separate transcripts. FIG. 26A is a schematic showing the AAV combinations injected in

each group two weeks before the crush injury and non-limiting exemplary expression

cassettes in Tet-Off systems encoding OCT4, SOX2, and/or KLF4. FIG. 26B is a chart

showing that expression of OSK from a single transcript improved axon regeneration relative

to expression of OCT4, SOX2, and KLF4 from separate transcripts. The number of

estimated axons per nerve after optic nerve crush injury as a function of the distance from the

site of injury (um) (µm) was quantified for mice receiving tTA virus and one of the following (1)

OCT4 virus, (2) SOX2 virus, (3) KLF4 virus, (4) virus with a vector encoding OCT4 and

SOX2 under one promoter (OCT4-SOX2), (5) separate OCT4, SOX2, and KLF4 viruses

(Oct4, Sox2, KLF4 or O,S,K), or (6) virus with a vector encoding OCT4, KLF4, and SOX2

under one promoter (Oct4-Sox2-KLF4 or OSK). The various vectors used are depicted in

FIG. 26A. FIG. 26C is a chart showing that expression of OSK from a single transcript

WO wo 2020/069373 PCT/US2019/053545

improved RGC survival relative to expression of OCT4, SOX2, and KLF4 from separate

transcripts. FIG. 26D includes whole mount staining of mouse retina showing that a

heterogeneous population of cells with few RBPMS+ cells were detected when separate viral

vectors encoding OCT4, SOX2, and KLF4 in separate viruses were delivered to the eye of

mice. Arrows point to seven different types of cells expressing OCT4, SOX2, KLF4, and/or

RBPMS in the upper left image under the schematic of the vectors. FIG. 26E includes data

showing that a more homogenous population of cells was detected when virus comprising a

viral vector encoding OSK under one promoter was delivered to the eye of mice as compared

to FIG. 26D. More OSK-expressing cells that were also RBPMS+ were detected as

compared to FIG. 26D. In the upper left image under the schematic of the vector used, the

long white arrow points to RBPMS+ cells expressing OCT4, SOX2, and KLF4 and the

shorter arrow indicates even some cells that did not express RBPMS expressed OSK.

[00171] FIG. 27 is a chart showing that Tet1 and Tet2 DNA demethylases play a role in

OSK-induced regeneration. The number of estimated axons per nerve after optic nerve crush

was quantified in mice receiving (1) OSK virus and a short hairpin control, (2) OSK virus

and a short hairpin against Tet1, or (3) OSK virus and a short hairpin against Tet2.

[00172] FIG.FIG. 28 includes datadata 28 includes showing thatthat showing expression of OSK expression using of OSK a Tet-Off using system a Tet-Off system

reversed age-related visual acuity loss in aged mice one month post injection of AAV virus

encoding TRE-OSK and AAV virus encoding tTA. FIG. 28 is a chart showing that

intravitreal injection of mice with virus encoding tTA and virus encoding TRE-OSK in the

absence of doxycycline (OSK induction condition) reversed the age-related decrease in the

spatial frequency threshold (cycles/degree, visual acuity test) observed in aged mice (12

month old (12m) and 18 month old (18m) mice). A visual acuity test based on optomotor

response (OMR) was used. As controls, age-matched mice received virus encoding virus

encoding rtTA and virus TRE-OSK in the absence of doxycycline (uninduced control). Adult

mice (3 month old (3m)) were also used as a control.

[00173] FIG. 29 includes data showing that expression of OSK reversed age-related

decline in retina ganglion cell (RGC) function in aged mice. FIG. 29 is a chart showing the

measurement of electrical waves generated from RGCs from adult (3 month old (3m)) and

aged (12 month old (12m) and 18 month old (18m)) mice. A pattern electroretinogram

(pattern ERG) was used. Mice were injected with rtTA virus and TRE-OSK virus without

doxycycline (uninduced control (ctl)) or with tTA virus and TRE-OSK virus (induced, OSK)

without doxycycline. Results were obtained one month after virus injection.

WO wo 2020/069373 PCT/US2019/053545

[00174] FIGS. 30A-30E include data showing that expression of OSK improved

glaucoma-induced declines in visual acuity and RGC function in one month-old mice. FIG.

30A is a chart showing that polystyrene microbeads induced glaucoma as measured by an

increase in intraocular pressure (IOP) compared to saline treatment in adult C57BL/6J mice.

The IOP measurements are shown in the first four weeks after microbead injection. FIGS.

30B-30C show that 4 weeks after microbeads injection into the anterior chamber of the eye,

there was significant loss of axon density and RGC density. AAVs were intravitreally

injected at 3 weeks post microbeads and it took another week before OSK expression was

observed. FIG. 30B includes a chart quantifying axon density (left panel) using p-

phenylenediamine (PPD) staining (shown, for example, on the right). FIG. 30C includes a

chart quantifying RGC cell density (left panel) using Brn3a staining (shown, for example, on

the right). FIG. 30D is a chart showing visual acuity improvement by OSK AAV treatment

in glaucoma-induced mice. Mice were intravitreally injected with microbeads to induce

glaucoma or saline without microbeads (no glaucoma control; saline). Three weeks after

microbeads injection, mice are then treated with (1) virus encoding rtTA and virus TRE-OSK

in the absence of doxycycline (beads (OSK AAV OFF)); (2) virus encoding tTA and virus

encoding TRE-OSK in the absence of doxycycline (beads (OSK AAV ON)). Results at 3

weeks after saline or microbead injection (pre-AAV injection) and 4 weeks after AAV

injection (7 week post microbeads) are shown. FIG. 30E is a chart showing RGC function

results by pattern electroretinogram in mice treated as in FIG. 30B.

[00175] FIGS.

[00175] FIGS. 31A-31C include 31A-31C include data data showing showingthat expression that of OSK expression of promoted neuronal OSK promoted neuronal

survival and axon regeneration of human SH-SY5Y neuronal cells following vincristine

(VCS)-induced damage. FIG. 31A includes a series of images showing the effect of inducing

OSK expression (OSK On) compared to no induction of OSK expression (OSK Off) on the

structure of neurons. Images were taken at day 3 and at day 9 after 24 hours of VCS

treatment. The outlines of the neuronal cell area are shown at Day 9. FIG. 31B is a chart

(um ² at indicated days after 24 hours of VCS treatment for cells quantifying neuron cell area (µm²)

in which OSK expression was induced (OSK On) and for cells in which OSK expression was

not induced (OSK Off). FIG. 31C is a chart quantifying neuron cell area (um ² at indicated (µm²)

days after 48 hours of VCS treatment for cells in which OSK expression was induced (OSK

On) and for cells in which OSK expression was not induced (OSK Off).

[00176] FIGs.

[00176] FIGs. 32A-32G show 32A-32G show that that partial partialreprogramming reprogrammingwith AAV-delivered with AAV-delivered

polycistronic OSK is non-toxic and induces CNS axon regeneration. FIG. 32A is a schematic

of the Tet-On and Tet-Off AAV vectors used in the study to control OSK expression. FIG.

WO wo 2020/069373 PCT/US2019/053545

32B shows body weight of WT mice, OSK transgenic mice, and AAV-mediated OSK-

(1.0x10¹² gene copies) with or without doxycycline induction in the first 4 expressing mice (1.0x1012

weeks (n=5, 3, 6, 4, 6, 3, respectively). FIG. 32C is a schematic showing intravitreal AAV

injection to target retina ganglion cells. Immunofluorescence of the whole-mounted display

and cross section of retina, showing the infection rate and targeted retina layer. The scale bars

represent 1 mm and 100 um, µm, respectively. FIG. 32D shows an experimental outline of the

optic nerve crush study using the Tet-Off system. FIG. 32E shows quantification of the

regenerating fibers by to d2EGFP, Oct4, Sox2, Klf4, OS, O+S+K, or OSK AAV at different

distances distal to to thethe lesion site.site. Error Error bars indicate s.e.m. (n=4-7). **** distances distal lesion bars indicate s.e.m. **** P<0.0001, (n=4-7). ANOVA , P<0.0001, ANOVA

with Bonferroni posttests. FIG. 32F shows the survival of RBPMS-positive cells in the RGC

*** layer transduced with different AAV vectors at day 14 post crush injury (n=4-8). ,

**** , P<0.0001, P<0.001, , P<0.0001, one-way one-way ANOVA ANOVA withwith Bonferroni Bonferroni post-tests, post-tests, relative relative to d2EGFP. to d2EGFP.

FIG. 32G shows representative images of optic nerve sections showing CTB-labeled axons in

wild-type mice with intravitreal injection of AAV2-tTA and TRE-OSK in the presence and

absence of DOX at 2 weeks after optic nerve injury. The crush site is indicated by asterisks.

The scale bars represent 200 um. µm.

[00177] FIGs.

[00177] FIGs. 33A-33K show 33A-33K show that that OSK OSK expression expressionpromotes axonaxon promotes regeneration and regeneration and

neuronal survival through a Tet-dependent mechanism. FIG. 33A shows experimental

strategies for pre- and post-injury induction of OSK expression. FIG. 33B shows RGC

survival in retinas with pre- and post-injury OSK expression. FIG. 33C shows the

quantification of regenerating fibers from pre-and pre- andpost-injury post-injuryOSK OSKexpression expressionmodels. models.FIG. FIG.

33D shows representative images of optic nerves showing regenerating axons at 4 weeks

after injury, with or without post injury OSK expression. The crush site is indicated with

asterisks. The scale bars represent 200 um. µm. FIGs. 33E-33F show the quantification of

regenerating fibers and RGC survival in retinas co-transduced with AAV2 vectors encoding

polycistronic OSK, tTA, and shRNA vectors with a scrambled sequence (Scr), Tet1, or Tet2

sequences toknockdown sequences to knockdown TetTet DNA DNA dioxygenases/demethylases. dioxygenases/demethylases. FIG. FIG. 33G 33G shows shows

experimental outlines for examining axon regeneration in human neurons post vincristine

(VCS) damage. FIG. 33H shows that OSK rejuvenates human neurons according to the skin

& blood clock. In the top panel of FIG. 33H, P value is calculated by linear regression model

to see if DNAmAge decrease with time. In the bottom panel of FIG. 33H, DNA methylation

age of human neurons with OSK expression pre (Day -) or after VCS damage (Day 1 and 9),

estimated by skin and blood cell clocks is shown. FIG. 33I shows the neurite area in each

AAV treatment AAV treatmentgroup. p < 0.05, group. 0.05,p << 0.01, 0.01,**** p <p 0.0001, one-way < 0.0001, ANOVA ANOVA one-way with Tukey's with Tukey's

WO wo 2020/069373 PCT/US2019/053545

multiple comparison test. FIG. 33J shows representative images of human neurons and the

neurite area after 9 days of recovery from VCS damage. FIG. 33K shows rDNA methylation

age of 1-month-old RGCs isolated from axon-intact retina infected with or without GFP, or

from axon-injured retinas infected with GFP-AAV or OSK-AAV 4 days after nerve crush.

[00178] FIGs.

[00178] FIGs. 34A-34H show 34A-34H show the the reversal reversalofofglaucoma by by glaucoma OSK OSK AAV AAV treatment. FIG. FIG. treatment.

34A is a schematic showing the experimental outline. FIG. 34B shows Intraocular pressure

measured weekly by rebound tonometry for the first 4 weeks post-microbead injection. FIG.

34C shows Representative micrographs of PPD-stained optic nerve cross-sections at 4 wks

post AAV2 or PBS injection. Scale bars, 50 um. µm. OSK Off (rtTA+TRE-OSK); OSK On

(tTA+TRE-OSK).FIG. (tA+TRE-OSK). FIG.34D 34Dshows showsaaquantification quantificationof ofhealthy healthyaxons axonsof ofthe theoptic opticnerve nerveat at44

weeks post PBS or AAV injection. FIG. 34E is a Schematic of High-contrast visual

stimulation assay to measure optomotor response. A reflexive head movement in response to

the rotation of a moving stripe pattern that increases in spatial frequency was used to assess

vision. FIG. 34F shows Spatial frequency threshold response of each mouse measured before

treatment and 4 weeks after intravitreal injection of AAV vectors. FIG. 34G shows

Representative pERG waveforms recorded from the same eye at baseline before treatment

and four weeks later after treatment with OSK-OFF AAV (top graph) or OSK-ON AAV

(bottom graph). FIG. 34H shows the Mean pERG amplitudes of recordings measured from

each mouse at baseline before treatment and 4 weeks after intravitreal injection of AAVs. *P

< 0.05; 0.05; <**P 0.01; ***p ***P < 0.01; < 0.001, ****P< <0.0001 < 0.001, 0.0001Two-way Two-wayANOVA ANOVAwith withTurkey Turkeyposttests posttests < between groups was used for the overall effect of time and treatment. A paired t-test was used

to compare before and after treatments.

[00179] FIGs.

[00179] FIGs. 35A-35I show 35A-35I show that that OSK OSK AAV AAVinduces inducesaxon regeneration axon and restores regeneration and restores

visual function in aged mice. FIG. 35A shows an Experimental outline for testing the effects

of OSK AAV treatment in aged mice on axon regeneration following optic nerve crush and

restoration of vision loss associated with physiological aging. FIG. 35B shows Axon

regeneration in 12-month-old mice with OSK AAV or control AAV (d2EGFP) treatment

following 2 or 5 weeks post optic nerve crush. FIG. 35C Representative confocal images of

longitudinal sections through the optic nerve showing CTB-labeled axons after 5 weeks of

OSK treatment. Scale bar represents 200 um. µm. FIG. 35D The spatial frequency threshold in

young mice (4 months) and old mice (12 months) treated with OSK-Off or OSK-On

AAVs.FIGs. 35E-35F show Spatial frequency threshold and pERG amplitudes in old mice

(12 months) treated with: (i) OSK-Off, (ii) OSK-On, or (iii) OSK-On plus either: sh-Scr, sh-

Tet1- or sh-Tet2-mediated knockdown of DNA demethylases. OSK-Off, (rtTa+TRE-OSK); (rtTA+TRE-OSK);

WO wo 2020/069373 PCT/US2019/053545

OSK-On, (tTA+OSK). FIG. 35G is hierarchical clustered heatmap showing RNA-Seq

expression of 464 differentially expressed genes in cell sorted purified RGCs from intact

young mice (5 months) or intact old mice (12 months), or old mice treated with either control

AAV (TRE-OSK) or OSK-On AAV. FIG. 35H is a scatter plot of OSK-induced changes in

RNA levels versus age-associated changes in mRNA levels. Dots represent differentially

expressed genes in RGCs. FIG. 35I shows rDNA methylation age of 12-month-old RGCs

FACS isolated from retinas infected for 4 weeks with -OSK or +OSK AAV together with

short-hairpin DNAs with a scrambled sequence (sh-Scr) or targeted to Tet1 or Tet2 (sh-

Tet1/sh-Tet2). Gene exclusion criteria for FIG. 35G and FIG. 35H: genes with low overall

expression (log2(CPM)<2), genes that did not significantly change with age (absolute log2

fold-change <1) or genes altered by the virus (differentially expressed between intact old and

old treatd with TRE-OSK AAV). * *PP<<0.05; 0.05;**P **P<<0.01; 0.01;<***P < 0.001, 0.001, P<0.0001. < 0.0001.

Two-way ANOVA in FIG. 35D; One-way ANOVA in FIGs. 35B, 35E and 35F.

[00180] FIGs. 36A-36H show an exploration of OSK (no Myc) effects on ageing and the

safety of OSK AAV. FIG. 36A is a schematic of an experimental outline of testing

reprogramming effect in young and old transgenic mouse fibroblasts. FIG. 36B shows OSKM

expression rescues age-associated transcriptional changes without inducing pluripotency. For

example, Nanog expression is not induced. FIG. 36C shows OSK expression rescues age-

associated transcriptional changes without inducing pluripotency. For example, Nanog

expression is not induced. FIG. 36D shows OSK AAV9 expression in the liver compared to

transgenic mice. FIG. 36E shows the body weight of WT mice and AAV-mediated OSK-

expressing mice (1.0x10^12 gene copies total) with or without doxycycline in the following 9

months after first 4 weeks (n=5, 3, 6, 4, respectively). FIG. 36F shows AAV-UBC-rtTA and

AAV-TRE-Luc vectors used for measuring tissue distribution. FIG. 36G shows luciferase

imaging of WT mice at 2 months after retro-orbital injections of AAV9-UBC-rtTA and AAV9-UBC-rtA and

AAV9-TRE-Luc (1.0x10^12 gene copies total). Doxycycline was delivered in drinking water

(1 mg/mL) for 7 days to the mouse shown on the right. FIG. 36H shows luciferase imaging of

eye (Ey), brain (Br), pituitary gland (Pi), heart (He), thymus (Th), lung (Lu), liver (Li),

kidney (Ki), spleen (Sp), pancreas (Pa), testis (Te), adipose (Ad), muscle (Mu), spinal cord

(SC), stomach (St), small intestine (In), and cecum (Ce) 2 months after retro-orbital injection

of AAV9-UBC-rtTA and AAV9-TRE-Luc followed by treatment with doxycycline for 7

days. The luciferase signal is primarily in liver. Imaging the same tissues with a longer

exposure time (FIG. 36H, lower panel) revealed lower levels of luciferase signal in pancreas

(liver was removed).

WO wo 2020/069373 PCT/US2019/053545

[00181] FIGs. 37A-37D show the characterization of an inducible polycistronic AAV

system. FIG. 37A shows an Immunofluorescence analysis of the whole-mounted retina

transduced with a polycistronic AAV vector expressing OCT4, SOX2, and KLF4 in the same

cell. Arrows point at triple positive cells. FIG. 37B shows an immunofluorescence analysis of

the whole-mounted retina transduced with AAVs separately encoding OCT4, SOX2, and

KLF4. Dotted arrows point to double-positive cells. Solid arrows point at single-positive

cells, except for arrow in lower right corner of each image, which points at a triple positive

cell. FIGs. 37C-37D are images showing whole-mounted retina display of RBPMS and Klf4

immunofluorescence. FIG. 37C shows that expression from AAV2 Tet-Off system can be

turned off by Dox drinking water (2mg/mL 3 days). FIG. 37D shows that expression from

AAV2 Tet-On system can be turned on by Dox drinking water (2mg/mL 2 days). Scale bars

represent 1mm.

[00182] FIGs. 38A-38C show that OSK induces long-term axon regeneration post injury

without RGC proliferation. FIG. 38A shows retina whole-mount staining showing OSK

infected RGCs have no proliferation marker Ki67 (left), while proliferating 293T cells have

Ki67 signal (right). The scale bars represent 100 um. µm. FIG. 38B shows whole nerve imaging

of optic nerves showing regenerating axons from control (no AAV) or OSK AAV treatment

at 3 months after injury. The scale bars represent 200 um. µm. FIG. 38C shows whole nerve

imaging showing CTB-labeled regenerative axons at 16 weeks post-injury (wpc) in wild-type

mice with intravitreal injection of AAV2-tTA and TRE-OSK. Scale bars represent 200 um. µm.

[00183] FIGs. FIGs. 39A-39D 39A-39D showshow Tet-On Tet-On system system has has better better turnturn on rate on rate and and OSK OSK transduced transduced

RGCs have higher survival rate. FIG. 39A shows Representative images showing the

d2EGFP expression in retina from Tet-Off AAV system with different Dox treatment. When

pre-treated with DOX to suppress expression (on DOX), the GFP expression only showed up

sparsely after DOX been withdrawal for 8 days, much weaker compared to peak expression

(Never DOX). FIG. 39B are representative images showing the d2EGFP in retina from Tet-

On AAV system. No GFP expression was observed in the absence of DOX, and GFP

expression reached peak in 2 days after Dox induction and didn't get stronger with 5 days of

DOX induction. FIG.39C shows representative Immunofluorescence image of GFP-positive

or KLF4-positive RGCs in intact and crushed samples. FIG.39D shows quantification of

GFP- GFP- or or KLF4- KLF4- positive positive cells cells indicating indicating higher higher survival survival rate rate of of OSK OSK expressing expressing RGCs RGCs after after

crush. Scale bars represent 200 um in FIG. 39A, FIG. 39B, and FIG. 39C.

[00184] FIGs. 40A-40F show identification of epigenetic mechanism underlying OSK

effect. FIG. 40A Representative images of retinal whole mounts transduced with d2EGFP- or

OSK-encoding AAV2 in the presence or absence of crush injury. The retinal whole mounts

were immunostained for RGC marker RBPMS and mTOR activation marker pS6. FIG. 40B

shows the quantification of pS6 positive RGC % in intact and crushed samples. FIG. 40C

quantification of transduction rate of shRNA-YFP AAV in the OSK expressed RGCs. FIG.

40D shows representative images of retinal whole mounts transduced with OSK-encoding

AAV2 in the combination with sh-Scr, sh-Tet1 or sh-Tet2 YFP AAV. The retinal whole

mounts were immunostained for Klf4. Scale bars represent 100 um µm in FIG. 40A and FIG.

40D. FIG. 40E shows Tet1 versus GAPDH mRNA level with sh-Scr or sh-Tet1 treatment in

mouse RGCs in the presence of OSK expression. FIG. 40F shows Tet2 versus GAPDH

mRNA level with sh-Scr or sh-Tet2 AAV in mouse RGCs in the presence of OSK

expression.

[00185] FIGs.

[00185] FIGs. 41A-41K 41A-41K show show that that OSKOSK robustly robustly induces induces human human neuron neuron axon axon

regeneration independent of mTOR pathway. FIG. 41A shows immunofluorescence of

differentiated human neurons with transduction of AAV-DJ vectors encoding TRE-OSK and

tTA (OSK On) or TRE-OSK alone (OSK Off). FIG. 41B shows mRNA level of Oct4, Sox2

and Klf4 of human neurons transduced with AAV-DJ vectors as in FIG. 41A. FIG. 41C

shows the FACS profile of G1, S, and G2 phases in undifferentiated cells and differentiated

cells with OSK On or Off. FIG. 41D shows the quantification of cell population that are in

proliferating S phase. FIG. 41E shows representative images and the neurite area of human

neurons post vincristine damage with or without OSK expression. FIG. 41F shows the

quantification of neurite area at different time points post vincristine damage. FIG. 41G

shows Tet2 mRNA level with sh-Scr and sh-Tet2 AAV treatment in human neurons. FIG.

41H shows the phosphorylation level of S6 in human neurons with Rapamycin treatment

(10nM) for 5 days. FIG. 41I shows the effect of mTOR inhibition on axon regeneration of

differentiated neurons with OSK Off or OSK On. FIG. 41J shows DNA methylation age of

human neurons before vincristine (VCS) damage (Day -) or 1 and 9 days post-damage in the

absence of OSK expression, estimated using a skin or a blood cell clock. FIG. 41K shows

mouse Oct4 mRNA level with sh-Scr or sh-Tet2 AAV in human neurons in the absence or

presence of OSK expression.

[00186] FIGs.

[00186] FIGs. 42A-42C show 42A-42C show the the effect effectofofOSK OSKinin a Microbead-induced mousemouse a Microbead-induced model.model.

FIG. 42A shows the quantification of RGCs and representative confocal microscopic images

from retinal flat-mounts stained with anti-Brn3a an RGC-specific marker, and DAPI, a

nuclear stain, at 4 weeks post-microbead or post-saline injection. The scale bar represents 75

mm. FIG. 42B shows the quantification of healthy axons of optic nerve and representative

WO wo 2020/069373 PCT/US2019/053545

photomicrographs of PPD-stained optic nerve cross-sections, at 4 weeks post-microbead or

post-saline injection. The scale bars represent 10 um. µm. FIG. 42C shows the quantification of

RGCs and representative confocal microscopic images at 4 weeks post AAV injection and 8

weeks post-microbead or post-saline injection.

[00187] FIG.FIG. 43A- show 43A-43G 43G show the effect the effect of in of OSK OSKaged in aged mice.mice. FIG. FIG. 43A shows 43A shows the effect the effect

of OSK expression on RGC survival in young, adult, and aged mice after optic nerve crush.

FIG. 43B shows the axon regeneration promoted by OSK expression compared to the

d2EGFP controls in young (1 month old), adult (3 months old), and aged (12 months old)

mice at 2 weeks post injury. FIG. 43C shows a comparison of pERG measurements in

different ages at one month post OSK off or OSK On treatment. OSK Off, rtTA+TRE=OSK

AAV; OSK On, tTA+OSK AAV. FIG. 43D shows comparison of RGC cell desnity in 4m-

and 12m-old mice at one month post OSK off or OSK On treatment. FIG. 43E shows a

comparison of axon density in 4m- and 12m-old-mice at one month post OSK off or OSK On

treatment. FIG. 43F shows comparison of pERG measurement in different ages at one-month

after -OSK or +OSK treatment. -OSK: AAV-rtTA+AAV-TRE-OSK; +OSK:

AAVtTA+AAV-TRE-OSK FIG. AAVtTA+AAV-TRE-OSK. FIG. 43G 43G shows shows spatial spatial frequency frequency threshold threshold in in 18-month-old 18-month-old

mice treated with -OSK or +OSK AAV for 4 weeks.

[00188] FIGs. 44A-44D FIGs. showshow 44A-44D RNA-seq analysis RNA-seq of genes analysis thatthat of genes reset their reset expression their by by expression

Reviver treatment. FIG. 44A is a scatter plot of OSK-induced changes in RNA levels versus

age-associated changes in mRNA levels. Dots represent differentially expressed genes in

RGCs are shown. Gene exclusion criteria: genes with low overall expression

(log2(CPM)<2), genes that did not significantly change with age (absolute log2 fold-change

<1) or genes altered by the virus (differentially expressed between intact old and old treatd

with TRE-OSK AAV). FIG. 44B is a hierarchical clustered heatmap showing RNA-Seq

expression of sensory genes in cell sorted purified RGCs from intact young mice (5 months)

or intact old mice (12 months), or old mice treated with either control AAV (TRE-OSK) or

OSK-On AAV. FIG. 44C shows the top 10 biological process that are lower in old compared

to young and restored by OSK. FIG. 44D shows the top 10 biological process that are higher

in old compared to young and reduced by OSK.

[00189] FIGs.

[00189] FIGs. 45A-45C show 45A-45C show methylation methylation clock clockanalysis of mouse analysis RGCs RGCs of mouse and human and human

neurons. FIG. 45A showscorrelation between rDNA methylation age and chronological age

of sorted mouse RGCs. FIG. 45B shows average DNA methylation levels of RGCs from

different ages and treatments. FIG. 45C shows average DNA methylation levels of human

WO wo 2020/069373 PCT/US2019/053545

neurons treated with OSK before treatment with vincristine (VCS) (-) or days post-VCS

damage (1 and 9).

[00190] FIGs. FIGs. 46A-46B 46A-46B show show that that OSK OSK mediates mediates axon axon regeneration regeneration in in aa Tet2-dependent Tet2-dependent

manner. A Tet2 conditional knockout mouse was used. Mouse eyes were injected with (1)

AAV-CRE (Tet2 cKO); (2) AAV-tTA + AAV-TRE-OSK: OSK (Tet2 WT); or (3) AAV-tTA

+ AAV-TRE-OSK + AAV-CRE: OSK (Tet2 cKO). Axon regeneration was assayed after

optic nerve crush. FIG. 46A are representative optic nerve images. FIG. 46B is a graph

quantifying axon numbers.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

[00191] The present disclosure is based, at least in part, on the unexpected results

demonstrating that expression of OCT4, SOX2, and KLF4 in the absence of exogenous C- c-

Myc expression can be used to promote partial reprogramming and tissue regeneration in

vivo. Surprisingly, using the eye as a model tissue, as described herein, in some

embodiments, it was determined that the combination of OCT4, SOX2, and KLF4 (OSK)

could be used to reset the youthful gene expression patterns and epigenetic age of retinal

ganglion cells to promote optic nerve regrowth and the restoration of vision in a rodent model

of glaucoma and in old animals. In some embodiments, the DNA demethylases Tet1 and

Tet2 are required for these restorative activities, which without being bound by a particular

theory, suggests that the DNA methylation clock is not just a correlate of age but a regulator

of it.

[00192] Provided herein, in certain embodiments, are engineered nucleic acids (e.g.,

expression vectors, including viral vectors) encoding OCT4, SOX2, and KLF4, each alone or

in combination, recombinant viruses (e.g., lentivirus, alphavirus, vaccinia virus, retrovirus,

adenovirus, herpes virus, or AAV) comprising the same, pharmaceutical compositions

comprising the engineered nucleic acids and/or recombinant viruses, kits comprising the

engineered nucleic acids and/or recombinant viruses, and methods of regulating (e.g.,

inducing, inducing and then stopping, etc.) cellular reprogramming, reversing aging, tissue

repair, organ regeneration, and tissue regeneration.

[00193] In certain embodiments, the expression of one of more of the genes is transient

(e.g., using an inducible promoter to regulate gene expression). Expression of one or more of

the genes (e.g., OCT4, SOX2, KLF4, or a combination thereof) may be modulated by altering

the activity of an inducing agent. As a non-limiting example, tetracycline transactivator

(tTA) is capable of inducing expression from a tetracycline-responsive promoter in the

WO wo 2020/069373 PCT/US2019/053545

absence of tetracycline. When tetracycline is added, tTA can no longer bind to the promoter

and induce cannot expression. As another non-limiting example, reverse tetracycline

transactivator (rtTA) is capable of inducing expression from a tetracycline-responsive

promoter in the presence of tetracycline. When tetracycline is removed, rtTA can no longer

bind to the promoter and cannot induce expression. As described herein, an inducible AAV

vector encoding OCT4, SOX2, and KLF4 (OSK) promoted optic regeneration in vivo

following damage. Therefore, the expression of these three genes may be useful in tissue and

organ regeneration, tissue and organ repair, reversing aging, treating neurodegenerative

diseases and conditions, cellular reprogramming, As described below, the vectors described

herein may be packaged, in some instances, into viruses with a titer of more than 2 X 1012 10¹²

particles per preparation and allow for precise control of OSK expression in mammalian cells

in vitro and in vivo.

[00194] Cellular Cellular reprograming reprograming allows allows for for the the production production of numerous of numerous cellcell types types fromfrom

existing somatic cells. Although the Yamanaka factors (OCT4, SOX2, KLF4 and c-Myc,

also known collectively as OSKM) have been shown to induce pluripotency in differentiated

cells, administration of these factors may induce teratomas or other cancers in vivo

(Takahashi et al., Cell. 2006 Aug 25;126(4):663-76); (Abad et al., Nature. 2013 Oct

17;502(7471):340-5). As a result of these safety concerns, use of the Yamanaka factors has

largely been limited to in vitro applications. Furthermore, existing methods of gene therapy

are plagued by inefficient and inconsistent gene transduction of target cells. The engineered

nucleic acids, recombinant viruses comprising the same, pharmaceutical compositions thereof

and kits provided herein overcome many of these limitations.

Engineered nucleic acids

[00195] The The engineered engineered nucleic nucleic acids acids of the of the present present disclosure disclosure may may encode encode OCT4, OCT4, SOX2, SOX2,

KLF4, and homologs or variants (e.g., functional variants) thereof, each alone or in

combination. In certain embodiments, an engineered nucleic acid (e.g., engineered nucleic

acid) does not encode c-Myc. In certain embodiments, an engineered nucleic acid (e.g.,

engineered nucleic acid) does not encode a functional c-Myc because it lacks a c-Myc

sequence. Assays to determine transcription factor (e.g., OCT4, SOX2, KLF4, or any

combination thereof) activity are known in the art and include cell-based transcription assays

and in vitro transcription assays. Transcription factor expression may also be determined

using other methods including enzyme-linked immunosorbent assays (ELISAs), western

blots, and quantification of RNA (e.g., using reverse transcription polymerase chain reaction).

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

A transcription

[00196] A transcription factor factor (e.g., (e.g., OCT4, OCT4, SOX2, SOX2, KLF4, KLF4, or homologs or homologs or variants or variants thereof, thereof,

including mammalian OCT4, mammalian SOX2, and mammalian KLF4) may be encoded by

a single nucleic acid, or a single nucleic acid (e.g., engineered nucleic acid) may encode two

or more transcription factors (e.g., each operably linked to a different promoter, or both

operably linked to the same promoter). For example, in certain embodiments, a nucleic acid

(e.g., engineered nucleic acid) may encode OCT4; SOX2; KLF4; OCT4 and SOX2; OCT4

and KLF4; SOX2 and KLF4; or OCT4, SOX2, and KLF4, in any order.

[00197] In certain In certain embodiments, embodiments, an engineered an engineered nucleic nucleic acidacid (e.g., (e.g., engineered engineered nucleic nucleic acid) acid)

encodes an inducing agent (e.g., tTA or rtTA). In certain embodiments, a nucleic acid (e.g.,

engineered nucleic acid) may encode one or more transcription factors (e.g., one, two or three

transcription factors) and an inducing agent. In certain embodiments, an inducing agent is

encoded by a separate nucleic acid (e.g., engineered nucleic acid) that does not also encode a a transcription factor (e.g., OCT4, SOX2, or KLF4). In certain embodiments, an inducing

agent is encoded by a the nucleic acid (e.g., engineered nucleic acid) that also encodes a

transcription factor (e.g., OCT4, SOX2, and/or KLF4). In certain embodiments, an inducing

agent is encoded by a nucleic acid (e.g., engineered nucleic acid) that also encodes one or

more transcription factors selected from the group consisting of OCT4; SOX2; KLF4; and

any combinations thereof (e.g., OCT4; SOX2; KLF4; OCT4 and SOX2; OCT4 and KLF4;

SOX2 and KLF4; or OCT4, SOX2, and KLF4).

[00198] The The transcription transcription factors factors described described herein herein (e.g., (e.g., OCT4, OCT4, SOX2, SOX2, KLF4, KLF4, or any or any

combination thereof) or inducing agents may comprise one or more amino acid substitutions.

Variants can be prepared according to methods for altering polypeptide sequences known to

one of ordinary skill in the art such as those found in references which compile such methods,

e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current

Protocols in Molecular Biology, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., New

York. Conservative substitutions of amino acids include substitutions made amongst amino

acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;

(f) Q, N; and (g) E, D.

[00199] In certain In certain embodiments, embodiments, the the engineered engineered nucleic nucleic acids acids of the of the present present disclosure disclosure

comprise RNA (e.g., mRNA) and/or DNA. In some embodiments, the RNA and/or DNA is is

further modified. As a non-limiting example, an nucleic acid (e.g., engineered nucleic acid)

of the present disclosure, may be modified RNA (e.g., mRNA) encoding OCT4, KLF4,

SOX2, an inducing, or any combination thereof. See, e.g., Warren et al., Cell Stem Cell.

WO wo 2020/069373 PCT/US2019/053545

2010 Nov 5;7(5):618-30. As a non-limiting example, the engineered nucleic acids (e.g.,

RNA, including mRNA, or DNA) of the present disclosure may be formulated in a

nanoparticle for delivery. See, e.g., Dong et al., Nano Lett. 2016 Feb 10;16(2):842-8. In

some embodiments, the nanoparticle comprises acetylated galactose. See, e.g., Lozano-

Torres et al., J Am Chem Soc. 2017 Jul 5;139(26):8808-8811. In some embodiments, the

engineered nucleic acids (e.g., RNA, including mRNA, or DNA) is electroporated or

transfected into a cell. In certain embodiments, the engineered nucleic acids are delivered as

a naked nucleic acid (e.g., naked DNA or naked RNA).

[00200] In some In some embodiments, embodiments, an engineered an engineered nucleic nucleic acidacid thatthat is formulated is formulated in ain a

nanoparticle for delivery is not an AAV vector. Suitable vector backbones for formulation in

a nanoparticle include, but are not limited to, NANOPLASMIDTM NANOPLASMID vectors and vectors NTC and '8' NTC Series '8' Series

Mammalian Expression Vectors. Non-limiting examples of vector backbones for formulation

in a nanoparticle include NTC9385R and NTC8685. Without being bound by a particular

theory, NTC '8' Series Mammalian Expression Vectors may be useful as they are generally

cleared by cells within weeks. The NTC '8' Series Mammalian Expression Vector comprises

a CMV promoter, which can be operably linked to a sequence encoding OCT4, KLF4, SOX2,

or a combination thereof. Without being bound by a particular theory, the

NANOPLASMIDTM vector may NANOPLASMID vector may be beless lessimmunogenic thanthan immunogenic other vectors other and express vectors at a and express at a

higher level and may express for a long time, which could be useful in long-term expression

of an of an operably operablylinked nucleic linked acid. nucleic In some acid. embodiments, In some the NANOPLASMID embodiments, TM vector vector the NANOPLASMID

may be useful in long term expression of OCT4, KLF4, SOX2, or a combination thereof.

[00201] In some In some embodiments, embodiments, engineered engineered nucleic nucleic acids acids encoding encoding OSK OSK may may be useful be useful in in

making induced pluripotent stem cells). Without being bound by a particular theory,

modified RNA (e.g., mRNA) may have an advantage of minimal activation of innate immune

responses and limited cytotoxicity, thereby allowing robust and sustained protein expression.

In some embodiments, the RNA (e.g., mRNA) comprises modifications including complete

substitution of either 5-methylcytidine (5mC) for cytidine or pseudouridine (psi) for uridine.

[00202] In some embodiments, OCT4, KLF4, and/or SOX2 expression may be activated

using a CRISPR-activating system. In some embodiments, expression of one or more

transcription factors selected from the group consisting of OCT4, KLF4, SOX2, and

combinations thereof may be activated using a CRISPR-activating system. See, e.g., Liao et

al., Cell. 2017 Dec (14;171(7):1495-1507.e15; Liu et 14;171(7):1495-1507.e15: Liu et al., al., 2018, 2018, Cell Cell Stem Stem Cell Cell 22, 22, 1-10 1-10

February 1, 2018. In general, a CRISPR-activating system comprises an enzymatically dead

Cas9 nuclease (or nuclease-deficient Cas9 (dCas9)) fused to a transcription activation

WO wo 2020/069373 PCT/US2019/053545

complex (e.g., comprising VP64, P65, Rta, and/or MPH). Non-limiting examples of

sequences encoding VP64, P65, Rta, and/or MPH are provided below. A VP64, P65, Rta, or

MPH may be encoded by a sequence that comprises a sequence that is at least 70% (e.g.,

75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to any of the VP64, P65, Rta,

and/or MPH sequences described herein. This Cas9 fusion protein may be referred to as a

CRISPR activator. A guide RNA targeting the promoter and/or enhancer region of a gene of

interest is used in a CRISPR-activating system to target the dCas9-transcription activation

complex and drive expression of the endogenous gene.

[00203] In In

[00203] someembodiments, some embodiments, expression expression ofofOCT4; KLF4; OCT4; SOX2; KLF4; or any SOX2; or combination any combination

thereof may be activated using a transcription activator-like effector nucleases (TALEN) or a

Zinc-finger nuclease (ZFN) system.

[00204] The The engineered engineered nucleic nucleic acids acids of the of the present present disclosure disclosure may may encode encode sgRNA sgRNA to to

target and the promoter and/or enhancer region of the endogenous locus of OCT4, SOX2,

and/or KLF4 in a cell. The engineered nucleic acids of the present disclosure may encode

sgRNA to target and the promoter and/or enhancer region of the endogenous locus of one or

more transcription factors selected from OCT4; SOX2; KLF4; and any combinations thereof

in a cell. In some embodiments, the engineered nucleic acid (e.g., expression vector) further

encodes a dCas9 (dead Cas9) and a transcriptional activation complex (e.g., VP64, P65, Rta,

MPH). In some embodiments, the dCas9 (dead Cas9) and a transcriptional activation

complex (e.g., VP64, P65, Rta, MPH) is administered to a cell on a engineered nucleic acid

(e.g. expression vector). In some embodiments, the vector encoding the sgRNA and/or a

dCas9 (dead Cas9) and a transcriptional activation complex (e.g., VP64, P65, Rta, MPH) is a

viral vector (e.g., AAV vector). In some embodiments, dCas9 (dead Cas9) and a

transcriptional activation complex (e.g., VP64, P65, Rta, MPH) is introduced into a cell as

protein.

[00205] In some In some embodiments, embodiments, guide guide RNA RNA targeting targeting the the enhancer enhancer and/or and/or promoter promoter region region

of OCT4, SOX2, and/or KLF4 is formulated in a nanoparticle and injected with dCas9-VP64

protein. In some embodiments, guide RNA targeting the enhancer and/or promoter region of

OCT4, SOX2, KLF4, or any combination thereof is formulated in a nanoparticle and injected

with dCas9-VP64 protein. In some embodiments, the guide RNA and/or nucleic acid

encoding dCas9 (dead Cas9) and a transcriptional activation complex (e.g., VP64, P65, Rta,

MPH) is administered as naked nucleic acid (e.g., naked DNA formulated in a nanoparticle).

In some embodiments, the guide RNA and/or nucleic acid encoding dCas9 (dead Cas9) and a

transcriptional activation complex (e.g., VP64, P65, Rta, MPH) is delivered via a

WO wo 2020/069373 PCT/US2019/053545

recombinant virus (e.g., lentivirus, adenovirus, retrovirus, herpes virus, alphavirus, vaccinia

virus or adeno-associated virus (AAV)).

[00206] Non-limiting example, sequences of guide RNAs targeting the endogenous OCT4

locus or SOX2 locus are provided in Liu et al., Cell Stem Cell. 2018 Feb 1;22(2):252-

261.e4. Non-limiting examples of guide RNAs targeting OCT4, SOX2, and/or KLF4 are also

provided in Weltner et al., Nat Commun. 2018 Jul 6;9(1):2643.

Without

[00207] Without being being bound bound by abyparticular a particular theory, theory, use use of aofCRISPR-CAS9 a CRISPR-CAS9 system system to to

activation endogenous expression of OCT4, KLF4, and/or SOX2 in the absence of c-Myc

expression may obviate potential toxicity associated with exogenous gene expression and/or

superphysiological gene expression.

[00208] Nucleic acids (e.g., engineered nucleic acids) encoding a transcription factor

(OCT4, SOX2, KLF4, or any combination thereof) or encoding an inducing agent) may be

introduced into an expression vector using conventional cloning techniques. Suitable

expression vectors include vectors with a promoter (e.g., a constitutive or inducible promoter,

including a TRE promoter) operably-linked to a nucleic acid (e.g., engineered nucleic acid)

encoding OCT4, SOX2, KLF4, or any combination thereof, and a terminator sequence (e.g., a

SV40 sequence as described herein). In some embodiments, a nucleic acid (e.g., engineered

nucleic acid) encodes a promoter operably linked to a nucleic acid encoding an inducing

agent. In some embodiments, a vector comprises a WPRE sequence. Expression vectors

containing the necessary elements for expression are commercially available and known to

one of ordinary skill in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory

Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012).

[00209] Vectors of the invention may further comprise a marker sequence for use in the

identification of cells that have or have not been transformed or transfected with the vector,

or have been reprogrammed. Markers include, for example, genes encoding proteins that

increase or decrease either resistance or sensitivity to antibiotics (e.g., ampicillin resistance

genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes

and chloramphenicol resistance genes) or other compounds, genes encoding enzymes with

activities detectable by standard assays known in the art (e.g., B-galactosidase, ß-galactosidase, senescence-

associated beta-galactosidase, luciferase or alkaline phosphatase), and genes that visibly

affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g.,

green fluorescent protein). In some embodiments, the vectors used herein are capable of

autonomous replication and expression of the structural gene products present in the DNA

segments to which they are operably linked.

WO wo 2020/069373 PCT/US2019/053545

[00210] In certain embodiments, the expression vector comprises an inducible promoter

(e.g., a tetracycline-responsive promoter) operably linked to a sequence encoding a

transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof). In certain

embodiments, the promoter operably linked to a sequence encoding a transcription factor

(e.g., OCT4, SOX2, KLF4, or any combination thereof) is a tissue-specific or cell type-

specific promoter (e.g., brain-specific, liver-specific, muscle-specific, nerve cell-specific,

glial cell-specific, endothelial cell-specific, lung-specific, heart-specific, bone-specific,

intestine-specific, skin-specific promoters, or eye-specific promoter). As an example, the

muscle-specific promoter may be a desmin promoter (e.g., a sequence that is at least 70%

(e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 29).

In S ome embodiments, an eye-specific promoter may be a promoter that is at least 70% (e.g.,

at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence selected

from SEQ ID NOs: 101-104.

[00211] In certain embodiments, the promoter operably linked to a sequence encoding a

transcription factor (e.g., OCT4, SOX2, KLF4, or any combination thereof) is age- or

senescence-specific (e.g. the age- or senescence-specific promoter may be a p16 promoter or

a Cas9-directed transcription factor that binds to methylated DNA, which is known to

accumulate with age).

[00212] In certain embodiments, an expression vector comprises a constitutive promoter

operably linked to a nucleic acid (e.g., engineered nucleic acid) encoding OCT4, SOX2,

KLF4, or any combination thereof. In some embodiments, such a vector may be inactivated

using a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/guide RNA

system. For example, a guide RNA may be complementary to the vector and is capable of

targeting a Cas9 nuclease to the vector. In some embodiments, the guide RNA is

complementary to a transgene (e.g. transgene encoding OCT4, KLF4, SOX2, or a

combination thereof) in any of the expression vectors described herein. Cas9 may then

generate double-stranded breaks in the vector and/or mutate the vector, rendering the vector

inactive. inactive.

In certain

[00213] In certain embodiments, embodiments, the the promoter promoter operably operably linked linked to atosequence a sequence encoding encoding an an

inducing agent is a constitutive promoter (e.g., CMV, EF1 alpha, a SV40 promoter, PGK1,

UBC, CAG, human beta actin gene promoter, or UAS). In certain embodiments, the

promoter operably linked to a sequence encoding an inducing agent is a tissue-specific

promoter (e.g., brain-specific, liver-specific, muscle-specific, nerve cell-specific, lung-

specific, heart-specific, bone-specific, intestine-specific, skin-specific promoters, or eye- specific promoter). As an example, the muscle-specific promoter may be a desmin promoter

(e.g., a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or

100%) identical to SEQ ID NO: 29).

A nucleic

[00214] A nucleic acid acid (e.g.,engineered (e.g., engineered nucleic nucleicacid) (e.g., acid) an expression (e.g., vector) an expression may vector) may

further comprise a separator sequence (e.g., an IRES or a polypeptide cleavage signal).

Exemplary polypeptide cleavage signals include 2A peptides (e.g., T2A, P2A, E2A, and

F2A). A 2A peptide may comprise a sequence that is at least 70% (e.g., at least 75%, 80%,

85%, 90%, 95%, 98%, 99%, or 100%) identical to SEQ ID NO: 9. For nucleic acids (e.g.,

engineered nucleic acids) (e.g., expression vectors) encoding more than one transcription

factor (e.g., OCT4, SOX2, KLF4, or any combination thereof), each transcription factor may

be operably linked to a different promoter or to the same promoter. The transcription factors

may be separated (e.g., by peptide separator sequence) on the nucleic acid. Expression of the

nucleic acid (e.g., engineered nucleic acid) results in separate amino acid sequences encoding

each transcription factor.

[00215] In certain embodiments, an expression vector (e.g., an expression vector encoding

OCT4, KLF4, SOX2, or a combination thereof) of the present disclosure may further

comprise a selection agent (e.g., an antibiotic, including blasticidin, geneticin, hygromycin B,

mycophenolic acid, puromycin, zeocin, actinomycin D, ampicillin, carbenicillin, kanamycin,

and neomycin) and/or detectable marker (e.g., GFP, RFP, luciferase, CFP, mCherry,

DsRed2FP, mKate, biotin, FLAG-tag, HA-tag, His-tag, Myc-tag, V5-tag, etc.).

[00216] In certain In certain embodiments, embodiments, an expression an expression vector vector encoding encoding an inducing an inducing agent agent of the of the

present disclosure may further comprise a selection agent (e.g., an antibiotic, including

blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin, actinomycin D,

ampicillin, carbenicillin, kanamycin, and neomycin) and/or detectable marker (e.g., GFP,

RFP, luciferase, CFP, mCherry, DsRed2FP, mKate, biotin, FLAG-tag, HA-tag, His-tag, Myc-

tag, V5-tag, etc.).

[00217] In certain In certain embodiments, embodiments, an expression an expression vector vector (e.g., (e.g., encoding encoding OCT4, OCT4, SOX2, SOX2,

KLF4, or any combination thereof) is present on a viral vector (e.g., AAV vector). In certain

embodiments, an expression vector encoding an inducing agent is present on a viral vector

(e.g., AAV vector). An AAV vector, as used herein, generally comprises ITRs flanking an

expression cassette (e.g., a nucleic acid (e.g., engineered nucleic acid) comprising a promoter

sequence operably linked to a sequence encoding OCT4, SOX2, KLF4, or any combination

thereof and a terminator sequence, a nucleic acid (e.g., engineered nucleic acid) comprising a

PCT/US2019/053545

promoter sequence operably linked to a sequence encoding an inducing agent, or a

combination thereof).

In certain

[00218] In certain embodiments, embodiments, the the number number of base of base pairs pairs between between two two ITRsITRs in AAV in an an AAV

vector of the present disclosure is less than 5 kilobases (kb) (e.g., less than 4.9 kb, less than

4.8 kb, less than 4.7 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb,

less than 4.2 kb, less than 4.1 kb, less than 4 kb, less than 3.5 kb, less than 3 kb, less than 2.5

kb, less than 2 kb, less than 1.5 kb, less than 1 kb, or less than 0.5 kb). In certain

embodiments, an AAV vector with a distance of less than 4.7 kb between two ITRs is capable

of being packaged into virus at a titer of at least 0.5 x10^10 particle forming units per ml

(pfu/ml), at least 1x10^10 pfu/ml, at least 5 X 10^10 pfu/ml, at least 1 X 10^11 pfu/ml, at least

5 X x 10^11 pfu/ml, at least 1 X x 10^12 pfu/ml, at least 2 X 10^12 pfu/ml, at least 3 X 10^12

pfu/ml, at least 4 X 10^12 pfu/ml, at least 5 X x 10^12 pfu/ml, at least 6 10112 x10^12pfu/ml, pfu/ml,at atleast least

7 X 10^12 pfu/ml, at least 8 X 10^12 pfu/ml, at least 9 X x 10^12 pfu/ml, or at least 1 X 10^13

pfu/ml.

[00219] In certain embodiments, an expression vector of the present disclosure is at least 1

kilobase (kb) (e.g., at least 1kb, 2 kb, 3 kb, 4 kb, 5 kb, 6kb, 7 kb, 8 kb, 9 kb, 10 kb, 50 kb, or

100 kb). In certain embodiments, an expression vector of the present disclosure is less than

10 kb (e.g., less than 9 kb, less 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4

kb, less than 3 kb, less than 2 kb, or less than 1 kb).

Without

[00220] Without

[00220] being being bound bound by abyparticular a particular theory, theory, an expression an expression vector vector (e.g., (e.g., an AAV an AAV

vector) that encodes OCT4, SOX2, and KLF4 under one promoter results in more efficient

transduction of all three transcription factors in vivo compared to separate nucleic acids (e.g.,

engineered nucleic acids) encoding one or two of the transcription factors. In certain

embodiments, the infection efficiency of a recombinant virus (e.g., lentivirus, alphavirus,

vaccinia virus, retrovirus, adenovirus, herpes virus, or AAV) harboring a vector of the

present disclosure in cells (e.g., animal cells, including mammalian cells) is at least 20%

(e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at

least 90%, or 100%).

Recombinant Viruses Recombinant Viruses

Aspects

[00221] Aspects of of thethe present disclosure present disclosure provide providerecombinant viruses recombinant (e.g., viruses lentiviruses, (e.g., lentiviruses,

alphaviruses, vaccinia viruses, adenoviruses, herpes viruses, retroviruses, or AAVs). The

recombinant viruses (e.g., lentiviruses, alphaviruses, vaccinia viruses, adenoviruses, herpes

viruses, retroviruses, or AAVs) may harbor a nucleic acid (e.g., engineered nucleic acid)

WO wo 2020/069373 PCT/US2019/053545

(e.g., expression vector) encoding a transcription factor (e.g., OCT4, SOX2, KLF4, or any

combination thereof), or a combination thereof. In some embodiments, a recombinant virus

harbors a nucleic acid encoding at least two transcription factors selected from OCT4, SOX2,

and KLF4 (e.g., OCT4 and SOX2; KLF4 and SOX2; OCT4, KLF4, and SOX2; or OCT4 and

KLF4). In some embodiments, a recombinant virus harbors a nucleic acid encoding at least

three transcription factors selected from OCT4, SOX2, and KLF4 (e.g., OCT4, SOX2, and

KLF4). In some instances, a recombinant virus of the present disclosure comprises a nucleic

acid encoding an inducing agent.

[00222] In certain In certain embodiments, embodiments, recombinant recombinant virus virus is aisrecombinant a recombinant AAV.AAV. In some In some

embodiments, a recombinant AAV has tissue-specific targeting capabilities, such that a

transgene of the AAV will be delivered specifically to one or more predetermined tissue(s).

Generally, the AAV capsid is a relevant factor in determining the tissue-specific targeting

capabilities of an AAV. An AAV capsid may comprise an amino acid sequence derived from

AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and variants thereof. Non-limiting examples of the tissue-specificity of AAV serotypes are

provided in Table 1. An "X" "x" indicates that the indicated AAV serotype is capable of

delivering a transgene to a specific tissue.

Table 1. Non-limiting examples of AAV serotypes and their utility in specific tissues.

Relevant Tissue

Immune Immune Central System Nervous (T-cells,

Muscle Central System B-cells (e.g., (Blood- Nervous and AAV brain serotype Skeletal System Dendritic Liver Heart Muscle) Eye (CNS) barrier) Pancreas Cells) Eye Lung AAV1 X X X AAV2 AAV2 X x X X AAV3 AAV3 X x X x x AAV4 x X X AAV5 AAV5 x X X X AAV6 (e.g., AAV6.2) X X x x AAV7 AAV7 X X x AAV8 AAV8 X x x X X AAV9 AAV9 x X X x X X X X X x AAV10 (e.g.,

AAVrh10) AAVrh10) x X X X X X X x AAVDJ AAVDJ X x X AAVPHP.B X X

WO wo 2020/069373 PCT/US2019/053545

[00223] Recombinant AAVs comprising a particular capsid protein may be produced

using any suitable method. See, e.g., U.S. Patent Application Publication, US 2003/0138772,

which is incorporated herein by reference. AAV capsid protein sequences also known in the

art. See, e.g., Published PCT Application, WO 2010/138263, which is incorporated herein by

reference. Generally, recombinant AAV is produced in a host cell with the following

components: (1) a nucleic acid (e.g., engineered nucleic acid) sequence encoding an AAV

capsid protein or a fragment thereof, (2) a nucleic acid (e.g., engineered nucleic acid)

encoding a functional rep gene, (3) a recombinant AAV vector comprising AAV inverted

terminal repeats flanking a transgene (e.g., nucleic acids (e.g., engineered nucleic acids)

encoding OCT4, KLF4, SOX2, or a combination thereof), and (4) helper functions that allow

for packaging of the recombinant AAV vector into AAV capsid proteins. In some instances,

a recombinant AAV vector comprises a nucleic acid encoding an inducing agent. In certain

embodiments, the helper functions are introduced via a helper vector that is known in the art.

In some

[00224] In some instances,a asuitable instances, suitable host host cell cellline (e.g., line HEK293T (e.g., cells) HEK293T may bemay cells) used befor used for

producing a recombinant AAV disclosed herein following routine practice. One or more

expression vectors encoding one or more of the components described above may be

introduced into a host cell by exogenous nucleic acids (e.g., engineered nucleic acids), which

can be cultured under suitable conditions allowing for production of AAV particles. When

needed, a helper vector can be used to facilitate replication, to facilitate assembly of the AAV

particles, or any combination thereof. In certain embodiments, the recombinant AAV vector

is present on a separate nucleic acid (e.g., engineered nucleic acid) from the other

components (e.g., a nucleic acid (e.g., engineered nucleic acid) sequence encoding an AAV

capsid protein or a fragment thereof, a nucleic acid (e.g., engineered nucleic acid) encoding a

functional rep gene, and helper functions that allow for packaging of the recombinant AAV

vector into AAV capsid proteins. In certain embodiments, a host cell may stably express one

or more components needed to produce AAV virus. In that case, the remaining components

may be introduced into the host cell. The supernatant of the cell culture may be collected,

and the viral particles contained therein can be collected via routine methodology.

Methods of activating OCT4, SOX2, and KLF4, each alone or in combination, and

replacements thereof

Aspects

[00225] Aspects of of thethe present disclosure, present disclosure, in insome someembodiments, relate embodiments, to activating relate to activating

OCT4, SOX2, and KLF4, each alone or in combination, in a cell, tissue and/or organ. In

some embodiments, OCT4, SOX2, and KLF4, each alone or in combination, is activated in

WO wo 2020/069373 PCT/US2019/053545

the absence of c-Myc activation. The cell, tissue, and/or organ may be in vivo (e.g., in a

subject) or be ex vivo. As used herein, activation includes any nucleic acid (e.g., nucleic acid

comprising RNA, comprising DNA, or any combination thereof), protein, antibody, chemical

agent, or any combination thereof that is capable of increasing the biological activity of a

protein of interest (e.g., OCT4, SOX2, and/or KLF4). Biological activity (e.g., gene

expression, reprogramming ability, transcription factor activity, etc.) may be measured using

any routine method known in the art. In some embodiments, any nucleic acid (e.g., nucleic

acid comprising RNA, comprising DNA, or any combination thereof), protein, antibody,

chemical agent, or any combination thereof described herein replaces OCT4, SOX2and/or

KLF4. In some embodiments, any nucleic acid (e.g., nucleic acid comprising RNA,

comprising DNA, or any combination thereof), protein, antibody, chemical agent, or any

combination thereof described herein replaces OCT4, SOX2, KLF4, or any combination

thereof. In some embodiments, any of the nucleic acids (e.g., engineered nucleic acid)

encoding an inducing agent, engineered proteins encoding an inducing agent, chemical agents

capable of modulating (e.g., activating or inhibiting) the activity of an inducing agent, and/or

recombinant viruses encoding an inducing agent described herein is used to activate an

inducing agent.

Activation

[00226] Activation of OCT4, of OCT4, SOX2, SOX2, and and KLF4, KLF4, eacheach alone alone or combination or in in combination includes includes

increasing expression (e.g., RNA and/or protein expression) of OCT4, SOX2, and KLF4,

each alone or in combination. In some embodiments, the expression of OCT4, SOX2, and

KLF4, each alone or in combination is increased by at least 1%, 5%, 10%, 20%, 30%, 40%,

50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%,

or 1000% after administration of a nucleic acid (e.g., nucleic acid comprising RNA,

comprising DNA, or any combination thereof) encoding OCT4, SOX2, and/or KLF4, protein

encoding OCT4, SOX2, and/or KLF4, antibody capable of activating encoding OCT4, SOX2,

and/or KLF4, chemical agent capable of activating encoding OCT4, SOX2, and/or KLF4, or

any combination thereof to a cell, tissue, organ, and/or subject compared to before

administration. In some embodiments, the expression of OCT4, SOX2, and KLF4, each

alone or in combination is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,

70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%

after administration of a nucleic acid (e.g., nucleic acid comprising RNA, comprising DNA,

or any combination thereof) encoding OCT4, SOX2, KLF4, or any combination thereof,

protein encoding OCT4, SOX2, KLF4, or any combination thereof, antibody capable of

activating encoding OCT4, SOX2, KLF4, or any combination thereof, chemical agent

PCT/US2019/053545

capable of activating encoding OCT4, SOX2, KLF4, or any combination thereof, or any

combination thereof to a cell, tissue, organ, and/or subject compared to before administration.

[00227] Activation Activation of aofinducing a inducing agent agent includes includes increasing increasing expression expression (e.g., (e.g., RNA RNA and/or and/or

protein expression) of an inducing agent. In some embodiments, the expression of an

inducing agent, is increased by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,

80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% after

administration of a nucleic acid (e.g., nucleic acid comprising RNA, comprising DNA, or any

combination thereof) encoding the inducing agent, protein encoding the inducing agent,

chemical agent capable of modulating the activity of the inducing agent, or any combination

thereof to a cell, tissue, organ, and/or subject compared to before administration.

Expression

[00228] Expression may may be measured be measured by any by any routine routine method method known known in the in the art,art, including including

quantification of the level of a protein of interest (e.g., using an ELISA, and/or western blot

analysis with antibodies that recognize a protein of interest) or quantification of RNA (e.g.,

mRNA) levels for a gene of interest (e.g., using reverse transcription polymerase chain

reaction).

[00229] In addition In addition to the to the engineered engineered nucleic nucleic acids acids discussed discussed herein, herein, OCT4, OCT4, SOX2, SOX2, KLF4, KLF4,

alone or in combination may be activated in a cell, tissue, organ, and/or subject through the

use of engineered proteins. For example, protein encoding OCT4, SOX2, and/or KLF4 may

be generated (e.g., recombinantly or synthetically) and administered to a cell, tissue, organ,

and/or subject through any suitable route. For example, protein encoding one or more

combinations thereof may be generated (e.g., recombinantly or synthetically) and

administered to a cell, tissue, organ, and/or subject through any suitable route.

[00230]

[00230] In In someembodiments, some embodiments, activating activating expression expressionof of OCT4; SOX2; OCT4; KLF4;KLF4; SOX2; a a

replacement thereof; or any combination thereof from a tetracycline-inducible expression

vector comprises administering a tetracycline (e.g., doxycycline) to a cell, organ, tissue, or a

subject. As one of ordinary skill in the art would appreciate, the route of tetracycline

administration may be dependent on the type of cell, organ, tissue, and/or characteristics of a

subject. In some embodiments, tetracycline is administered directly to a cell, organ, and/or

tissue. As a non-limiting example, tetracycline may be administered to the eye of a subject

through any suitable method, including eye drops comprising tetracycline, sustained release

devices (e.g., micropumps, particles, and/or drug depots), and medicated contact lenses

comprising a tetracycline). In some embodiments, tetracycline is administered systemically

(e.g., through drinking water or intravenous injection) to a subject. Tetracycline may be

PCT/US2019/053545

administered topically (e.g., in a cream) or through a subcutaneous pump (e.g., to deliver

tetracycline to a particular tissue). Tetracycline may be administered intravenously,

intradermally, intraarterially, intralesionally, intratumorally, intracranially, intraarticularly,

intraprostaticaly, intrapleurally, intranasally, intravitreally, intravaginally, intrarectally,

topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctival,

bathing target cells directly, via a catheter, in creams, in particles (e.g., nanoparticles,

microparticles), in lipid compositions (e.g., liposomes), or by other method or any

combination of the forgoing as would be known to one of ordinary skill in the art (see, for

example, Remington's Pharmaceutical Sciences (1990), incorporated herein by reference).

[00231] As aAsnon-limiting a non-limiting example, example, an engineered an engineered protein protein may may be further be further modified modified or or

formulated for delivery to a cell, tissue, organ, and/or subject. For example, protein

transduction domains (i.e., PTD or cell-penetrating peptides) may be attached to an

engineered protein (e.g., OCT4, SOX2, and/or KLF4). As a non-limiting example, a protein

transduction domain (i.e., PTD or cell-penetrating peptide) may be attached to an engineered

protein encoding an inducing agent. Without being bound by a particular theory, a protein

transduction domain facilitate delivery of a cargo (e.g., a protein, nucleic acids, nanoparticles,

viral particles, etc.) across cellular membranes. Protein transduction domains include

cationic peptides, hydrophobic peptides, and/or a cell specific peptides. See, e.g., Zhou et al.,

Cell Stem Cell. 2009 May 8;4(5):381-4; Zahid et al., Curr Gene Ther. 2012 Oct;12(5):374-

80. 80.

[00232] In In

[00232] someembodiments, some embodiments, a protein proteinencoding encodingOCT4, SOX2, OCT4, and/or SOX2, KLF4,KLF4, and/or and/orand/or an an inducing agent is formulated in a nanoparticle (e.g., for nuclear delivery). In some

embodiments, a protein encoding OCT4, SOX2, KLF4, or any combination thereof (e.g.,

OCT4 and SOX2; KLF4 and SOX2; OCT4 and KLF4; or KLF4, SOX2, and OCT4) is

formulated in a nanoparticle (e.g., for nuclear delivery). In certain embodiments, a

nanoparticle further comprises a protein encoding an inducing agent. For example, chitosan

[poly(N-acetyl glucosamine)] is a biodegradable polysaccharide and may be used to

formulate nanoparticles by several methods. In some embodiments, a chitosan polymeric

nanoparticle is loaded with protein encoding OCT4, SOX2, and/or KLF4, and/or an inducing

agent and is delivered to the nucleus of a cell. See, e.g., Tammam et al., Oncotarget. 2016

Jun 21;7(25):37728-37739.

In some

[00233] In some embodiments, embodiments, a chemical a chemical agent, agent, antibody antibody and/or and/or protein protein replaces replaces OCT4, OCT4,

SOX2, and/or KLF4. In some embodiments, a chemical agent, antibody, a protein, or any

combination thereof replaces OCT4, SOX2, KLF4, or any combination thereof (e.g., OCT4

and SOX2; OCT4 and KLF4; KLF4 and SOX2; or KLF4, SOX2, and OCT4). For example,

a chemical agent, antibody and/or protein may promote expression of OCT4, SOX2, and/or

KLF4. In certain instances, a chemical agent, antibody and/or protein may promote

combinations thereof. In some embodiments, a chemical agent, antibody and/or protein may

activate target genes downstream of OCT4, SOX2, and/or KLF4. In some embodiments, a

chemical agent, antibody , a a protein, protein, oror any any combination combination thereof thereof may may activate activate target target genes genes

downstream of one or more transcription factors selected from the group consisting of OCT4;

SOX2; KLF4; and any combinations thereof. In some embodiments, a chemical agent,

antibody and/or protein is said to replace OCT4, SOX2, and/or KLF4 if the chemical agent,

antibody and/or protein may be used together with the other two transcription factors and

promote cellular reprogramming. In some embodiments, a chemical agent, antibody, protein,

or any combination thereof is said to replace OCT4, SOX2, KLF4, or any combination

thereof if the chemical agent, antibody, protein or any combination thereof may be used

together with the other two transcription factors and promote cellular reprogramming. For

example, cellular reprogramming may be determined by measuring gene expression (e.g.,

expression of embryonic markers and/or pluripotency markers). In some embodiments,

pluripotency markers include AP, SSEA1, and/or Nanog.

In some

[00234] In some embodiments, embodiments, an antibody an antibody is used is used to activate to activate OCT4, OCT4, SOX2, SOX2, and/or and/or

KLF4. In some embodiments, an antibody is used to activate one or more transcription

factors selected from OCT4, SOX2, KLF4, or any combination thereof. In some

embodiments, the antibody does not target OCT4, SOX2, and/or KLF4. In some

embodiments, the antibody does not target OCT4, SOX2, KLF4, or any combination thereof.

In some embodiments, the antibody increases expression of OCT4, SOX2, and/or KLF4. In

some embodiments, the antibody increases expression of OCT4, SOX2, KLF4, or any

combination thereof. In some embodiments, the antibody does not increase expression of

OCT4, SOX2, and/or KLF4. In some embodiments, an antibody replaces OCT4, SOX2,

and/or KLF4. In some embodiments, the antibody does not increase expression of OCT4,

SOX2, KLF4, or any combination thereof. In some embodiments, an antibody replaces

OCT4, SOX2, KLF4, or any combination thereof. Any suitable method of identifying

antibodies that can replace a transcription factor (e.g., OCT4, SOX2, and/or KLF4) may be

WO wo 2020/069373 PCT/US2019/053545

used. Any suitable method of identifying antibodies that can replace a transcription factor

(e.g., OCT4, SOX2, KLF4, or any combination thereof) may be used. See, e.g., Blanchard et

al., Nat Biotechnol. 2017 Oct;35(10):960-968.

In some

[00235] In some embodiments, embodiments, another another protein protein (e.g., (e.g., a nucleic a nucleic acidacid encoding encoding the the protein protein

or a polypeptide encoding the protein) may be used to replace OCT4, SOX2, and/or KLF4.

In some embodiments, another protein (e.g., a nucleic acid encoding the protein or a

polypeptide encoding the protein) may be used to replace OCT4, SOX2, KLF4, or a

combination thereof. For example, OCT4 may be replaced by Tet1, NR5A-2, Sall4, E-

cadherin, NKX3-1, or any combination thereof. In some embodiments, OCT4, SOX2, and/or

KLF4 may be replaced by NANOG and/or TET2. In some embodiments, OCT4, SOX2,

KLF4, or any combination thereof may be replaced by NANOG and/or TET2. See, e.g., Nat

Cell Biol. 2018 Aug;20(8):900-908; Gao et al., Cell Stem Cell. 2013 Apr 4;12(4):453-69.

Nanog and Lin28 can replace Klf4. See, e.g., Yu et al, Science. 318, 1917-1920, 2007). In

some embodiments, OCT4, SOX2, and/or KLF4 is replaced by Tet3 (tet methylcytosine

dioxygenase 3). In some embodiments, OCT4, SOX2, KLF4, or any combination thereof is

replaced by Tet3 (tet methylcytosine dioxygenase 3). In some embodiments, a nucleic acid

encoding a Tet1 DNA demethylase comprises a sequence that is at least 70% (e.g., at least

75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to NM_030625.3 or

NM_001253857.2. In some embodiments, an amino acid encoding a Tet1 DNA demethylase

99%, or 100%) identical to NP_085128.2 or NP_001240786.1. In some embodiments, a

nucleic acid encoding a Tet2 DNA demethylase comprises a sequence that is at least 70%

(e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to

NM_001127208.2, NM_001040400.2, NM_001346736.1, or NM_017628.4. In some

embodiments, an amino acid encoding a Tet2 DNA demethylase comprises a sequence that is

at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to

NP_060098.3, NP_001035490.2, NP_001333665.1, or NP_001120680.1. In some

embodiments, a nucleic acid encoding a Tet3 DNA demethylase comprises a sequence that is

NM_001287491.2, NM_001347313.1, NM_183138.2, or NM_001366022.1. In some

embodiments, an amino acid encoding a Tet3 DNA demethylase comprises a sequence that is

NP_001274420.1, NP_001334242.1, NP_898961.2, or NP_001352951.1. Tet1, Tet2, and/or

Tet3 may be derived from any species. In some embodiments, Tet1, Tet2, and/or Tet3 is a

WO wo 2020/069373 PCT/US2019/053545

truncated form of a wild-type counterpart. As a non-limiting example, Tet1, Tet2, and/or

Tet3 is N-terminally truncated compared to a wild-type Tet1, Tet2, and/or Tet3 counterpart

and is catalytically active. In some embodiments, Tet1, Tet2, and/or Tet3 only comprises the

catalytic domain of Tetl, Tet1, Tet2, and/or Tet3. In some embodiments, Tet1, Tet2, and/or Tet3

comprises the catalytic domain of Tet1, Tet2, Tet3, or any combination thereof. Non-limiting

examples of functional truncated Tet1 may be found in Hrit et al., Elife. 2018 Oct 16;7. pii:

e34870. e34870.

[00236] Additional Additional methods methods of replacing of replacing OCT4, OCT4, SOX2, SOX2, and/or and/or KLF4KLF4 to promote to promote cellular cellular

reprogramming are known in the art. See, e.g., Heng et al., Cell Stem Cell 6, 167-174

(2010); Eguchi et al., Proc. Natl Acad. Sci. USA 113, E8257-E8266 (2016); Gao et al., Cell

Stem Cell 12, 453-469 (2013); Long et al., Cell Res. 25, 1171-1174 (2015); Hou et al.,

Science 341, 651-654 (2013); Redmer et al., EMBO Rep. 12, 720-726 (2011); Tan et al., J.

Biol. Chem. 290, 4500-4511 (2014); Anokye-Danso et al., Cell Stem Cell 8, 376-388

(2011); Miyoshi et al., Cell Stem Cell 8, 633-638 (2011); Shu et al., Cell 153, 963-975

(2013); Yu, J. et al., Science 318, 1917-1920 (2007).

[00237]

[00237] In In someembodiments, some embodiments, a chemical chemicalagent agentreplaces OCT4, replaces SOX2, OCT4, and/or SOX2, KLF4 KLF4 and/or

(e.g., can be used in place of OCT4, SOX2, and/or KLF4 along with the other two

transcription factors to promote cellular reprogramming). In some embodiments, a chemical

agent replaces OCT4, SOX2, KLF4, or any combination thereof (e.g., can be used in place of

OCT4, SOX2, KLF4, or any combination thereof, along with the other two transcription

factors to promote cellular reprogramming). For example, SOX2 may be replaced by CHIR,

FSK, or 616452. OCT4 may be replaced by DZNep. Since Sall4 may be used to replace

OCT4 as mentioned above, any compound that replaces Sall4 may also be used to replace

OCT4. For example, CHIR, FSK, and 616452 may be used to replace Sall4. Nanog may be

replaced with 2i medium. See, e.g., Hou et al., Science. 2013 Aug 9;341(6146):651-4. See,

also, e.g., Zhao et al., Cell. 2015 Dec 17;163(7):1678-91.

In some

[00238] In some embodiments, embodiments, chemical chemical reprogramming reprogramming comprises comprises using using chemicals chemicals thatthat

reduce the toxicity of chemical agents that induce reprogramming. Non-limiting examples of

chemicals that reduce the toxicity of chemical reprogramming include ROCK inhibitors (e.g.,

Y27632 and Fasudil) and P38 MAPK inhibitors (e.g., SB203580 and BIRB796). See, e.g., Li

et al., Cell Stem Cell. 2015 Aug 6;17(2):195-203.

[00239] OCT4, KLF4, SOX2, replacements, or any combination thereof may be activated

(e.g., expression may be induced) in combination with activating an enhancer of

reprogramming and/or inhibiting a barrier of reprogramming. An enhancer of

WO wo 2020/069373 PCT/US2019/053545

reprogramming may be activated using any suitable method known in the art, including

overexpression of the enhancer, increasing expression of an endogenous gene encoding the

enhancer (e.g., using CRISPR technology), use of a chemical agent and/or antibody to

increase the biological activity of the enhancer, and use a chemical agent and/or antibody to

promote expression of the enhancer. A barrier of reprogramming may be inhibited using any

suitable method known in the art, including knocking down expression of the inhibitor (e.g.,

with siRNAs, miRNAs, shRNAs), knocking out an endogenous copy of the inhibitor (e.g.,

using CRISPR technology, TALENs, zinc finger nucleases, etc.), using a chemical agent

and/or antibody to decrease the biological activity of the inhibitor, and using a chemical agent

and/or antibody to decrease expression of the inhibitor.

[00240] Non-limiting examples of enhancers and barriers of reprogramming are provided

in Table 2. See also, e.g., Ebrahimi, Cell Regen (Lond). 2015 Nov 11;4:10, which is

incorporated by reference in its entirety for this purpose.

Table 2. Non-limiting examples of strategies to enhance reprogramming.

Reprogramming Enhancing Strategy Enhancers C/EBPa; UTF1; Mef2c; C/EBP; UTF1; Mef2c; Tdgf1; Tdgf1; FOXH1; FOXH1; GLIS1; GLIS1; mutated mutated reprogramming factors, MDM2; Bcl-2; CCL2; Kdm3a, Kdm3b, Kdm4c, and Kdm4b/2b; Jhdm1a/lb; Jhdm1a/1b; MOF; Mbd1-4 (or their small molecule activators); Wnt/B-catenin signaling; small molecule Pitstops 1 and 2;

Activation of vitamin C, palbiociclib; cytokines, e.g. IL-6; CDK4, CDK8, CDK19;

Enhancers lincU

Barriers Barriers p53, p57, p38, p16 k4a/p19Arf p21cipl, Rb p53, p57, p38, p21¹, Rb TGF-B, MAP kinase, Aurora A kinase, MEK/ERK, Gsk3, Wnt/B-catenin TGF-ß, signaling pathways, LATS2, PKC, IP3K, CDK8, CDK19. Native/somatic gene or transcriptional regulatory network (GRN/TRN). Specific members of ADAM family (e.g., ADAM7, ADAM21, ADAM29), endocytosis: (e.g., DRAMI, DRAM1, SLC17A5, ARSD), phosphatase: (e.g., PTPRJ, PTPRK, PTPN11). Inhibition of Chromatin regulators: (e.g., ATF7IP, MacroH2A, Mbd1-4, Setdbla. Barriers Barriers Transcription factors: (e.g., TTF1, TTF2, TMF1, T), Bright. Fbxw7 (a member of ubiquitin-proteasome system (UPS)) Lzts1, Ssbp3, Arx, Tfdp1, Nfe2, Ankrd22, Msx3, Dbx1, Lasp1, and Hspa8. Cytokines Cytokinese.g., e.g.,TNFa TNF Cells (e.g., senescent cells and NK cells) (e.g., navitoclax, BAY117082) NuRD, Mbd1-4, Gatad2a, Chd4 (see, e.g., Mor et al., Cell Stem Cell. 2018 Sep 6;23(3):412-425.e10)

WO wo 2020/069373 PCT/US2019/053545

KDM1a Kaiso (see, e.g., Kaplun et al., Biochemistry (Mosc). 2019 Mar;84(3):283-290) Mar;84(3):283-290)

Additional

[00241] Additional reprogramming reprogramming enhancers enhancers thatthat may may be activated be activated in combination in combination withwith

activation of OCT4, KLF4, SOX2, replacements thereof, or any combination thereof, include

histone lysine demethylases (e.g., KDM2, KDM3, and KDM4). Histone lysine demethylases

may be activated by being overexpressed in a cell, tissue, organ, and/or a subject. Chemical

activators of histone lysine demethylases are also encompassed by the present disclosure. For

example, vitamin C may be used to activate KDM3 and/or KDM4.

[00242] In In

[00242] someembodiments, some embodiments, OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,replacements thereof, replacements or any thereof, or any

combination thereof, is activated along with activation of C/EBPa andTfcp211. C/EBP and Tfcp211.Without Without

being bound by a particular theory, C/EBPa, and Tfcp211 C/EBP, and Tfcp211 together together with with Klf4 Klf4 may may drive drive Tet2- Tet2-

mediated enhancer demethylation and activation during reprogramming.

[00243] In In

[00243] someembodiments, some embodiments, OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,replacements thereof, replacements or any thereof, or any

combination thereof are activated in a cell, tissue, organ and/or a subject in combination with

a cytokine that facilitates reprogramming. IL6 is a non-limiting example of a cytokine. See,

e.g., Mosteiro et al, Science. 2016 Nov 25;354(6315), which is hereby incorporated by

reference in its entirety for this purpose.

[00244] In some embodiments, OCT4, SOX2, KLF4, replacements thereof, or any

activation of a miRNA (e.g., administration of a miRNA and/or expression of a miRNA).

For example, a miRNA that promotes cell cycle progression may be introduced to a cell,

tissue, organ, and/or subject. Non-limiting examples of miRNAs that promote cell cycle

progression include miR 302-367, miR 371-373, miR-200b, miR-200c, miR-205, miR 290-

295, miR-93, miR-106, and miR 135b.

[00245] As a non-limiting example, nerve regeneration may be enhanced by combining

activation of OCT4, SOX2, KLF4, replacements thereof, or any combination thereof with

activation of an enhancer. Non-limiting activation of enhancers include overexpression of a

member of the KLF family (e.g., KLF7), overexpression of c-Myc, STAT3 activation,

SOX11 overexpression, overexpression of Lin28, overexpression of or delivery of soluble

protein encoding insulin-like growth factor 1 (IGF1) and osteopontin (OPN), and activation

of B-RAF (e.g., introduction of a gain of function mutation). See also, e.g., Blackmore et al.,

Proc Natl Acad Sci USA. US A.2012 2012May May8;109(19):7517-22; 8;109(19):7517-22;Belin Belinet etal., al.,Neuron. Neuron.2015 2015May May

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

20;86(4):1000-1014; Bareyre et al., Proc Natl Acad Sci USA. 2011 Apr 12;108(15):6282-7;

Norsworthy et al., Neuron. 2017 Jun 21;94(6):1112-1120.e4; Wang et al., Cell Rep. 2018 Sep

4;24(10):2540-2552.e6; 4;24(10):2540-2552.e6; Liu Liu et et al., al., Neuron. Neuron. 2017 2017 Aug Aug 16;95(4):817-833; 16;95(4):817-833; O'Donovan O'Donovan et et al., al., JJ

Exp Med, 2014. 211(5): p. 801-14, which is each hereby incorporated by reference in its

entirety for this purpose.

[00246] In In

[00246] someembodiments, some embodiments, OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,replacements thereof, replacements or any thereof, or any

combination thereof, are activated in a cell, tissue, organ, and/or a subject in combination

with suppression or knockdown of reprogramming barriers. Non-limiting examples of

reprogramming barriers include Chafla, Chaflb, Ube2i, sumo2, and/or Nudt21. See, e.g.,

Brumbaugh et al., Cell. 2018 Jan 11;172(1-2):106-120.e21 11;172(1-2):106-120.e21;Cheloufi Cheloufiet etal., al.,Nature. Nature.2015 2015Dec Dec

10;528(7581):218-24; and Borkent et al., Stem Cell Reports, 2016. 6(5): p. 704-716, which is

each hereby incorporated by reference in its entirety for this purpose.

[00247]

[00247] As As a non-limiting example, a non-limiting example, aa reprogramming reprogrammingbarrier may may barrier be a be DNAa DNA

methyltransferase (DNMT) may be and a DNMT may be inhibited to promote

reprogramming of a tissue, cell, and/or organ. Most DNA methyltransferases use S-adenosyl-

L-methionine as a methyl donor. DNMT may be from any species. There are at least three

different types of methyltransferases. m6A methyltransferases are capable of methylating the

amino group at the c-6 position of adenines in DNA (e.g., Enzyme Commission (EC) No.

2.1.1.72). m4C methyltransferases are capable of generating N4-methylcytosine (e.g.,

Enzyme Commission (EC) No. 2.1.1.113). M5C methyltransferases are capable of

generating C5-methylcytosine (e.g., Enzyme Commission (EC) No. 2.1.1.37).

[00248] Non-limiting

[00248] Non-limiting examples examples of of mammalian mammalian DNADNA methyltransferases methyltransferases (DNMTs) (DNMTs) include include

DNMT1 and its isoforms DNMT1b and DNMT1o (oocytes-specific), DNMT3a, DNMT3b,

DNMT3L. GenBank Accession Nos. NM_001130823.3 (isoform a), NM_001318730.1

(isoform c), NM_001318731.1 (isoform d), and NM_001379.3 (isoform b) are non-limiting

examples of nucleotide sequences encoding human DNMT1. A nucleic acid encoding a

DNMT1 may comprise a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%,

95%, 98%, 99%, or 100%) identical to a sequence set forth in GenBank Accession Nos.

NM_001130823.3 (isoform a), NM_001318730.1 (isoform c), NM_001318731.1 (isoform d),

and/or NM_001379.3 (isoform b). GenBank Accession Nos. NP_001124295.1 (isoform a),

NP_001305659.1 (isoform c), NP_001305660.1 (isoform d), and NP_001370.1 (isoform b)

are non-limiting examples of amino acid sequences encoding human DNMT1. An amino

acid sequence encoding a DNMT1 may comprise a sequence that is at least 70% (e.g., at least

75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence set forth in

GenBank Accession Nos. NP_001124295.1 (isoform a), NP_001305659.1 (isoform c),

NP_001305660.1 (isoform d), and/or NP_001370.1 (isoform b). A nucleic acid encoding

human DNMT3A includes GenBank Accession No. NM_001320892.1, NM_001320893.1,

NM_022552.4, NM_153759.3, NM_175629.2, and NM_175630.1. A nucleic acid encoding

a DNMT3A may be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or

100%) identical to a sequence set forth in GenBank Accession Nos. NM_001320892.1,

NM_001320893.1, NM_022552.4, NM_153759.3, NM_175629.2, and/or NM_175630.1. An

amino acid sequence encoding human DNMT3A includes GenBank Accession Nos.

NP_001307821.1, NP_001307822.1, NP_072046.2, NP_715640.2, NP_783328.1, and

NP_783329.1. An amino acid sequence encoding a DNMT3A may be at least 70% (e.g., at

least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence set forth in

GenBank Accession Nos. NP_001307821.1, NP_001307822.1, NP_072046.2, NP_715640.2,

NP_783328.1, and/or NP_783329.1. A nucleic acid encoding human DNMT3B includes

GenBank Accession No. NM_001207055.1, NM_001207056.1, NM_006892.3, NM_001207056.1 NM_006892.3,

NM_175848.1, NM_175849.1, and NM_175850.2. A nucleic acid encoding a DNMT3B

may be at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical

to a sequence set forth in GenBank Accession Nos. NM_001207055.1, NM_001207056.1,

NM_006892.3, NM_175848.1, NM_175849.1, and/or NM_175850.2. An amino acid

sequence encoding human DNMT3B includes GenBank Accession Nos. NP_001193984.1,

NP_001193985.1, NP_008823.1, NP_787044.1, NP_787045.1, and NP_787046.1. An amino

acid sequence encoding a DNMT3B may be at least 70% (e.g., at least 75%, 80%, 85%, 90%,

NP_001193984.1, NP_001193985.1, NP_008823.1, NP_787044.1, NP_787045.1, and/or

NP_787046.1. A nucleic acid encoding human DNMT3L includes GenBank Accession No.

NM_013369.3 and NM_175867.2. A nucleic acid encoding a DNMT3L may be at least 70%

(e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%) identical to a sequence set

forth in GenBank Accession Nos. NM_013369.3 and/or NM_175867.2. An amino acid

sequence encoding human DNMT3L includes GenBank Accession Nos. NP_037501.2 and

NP_787063.1. An amino acid sequence encoding a DNMT3L may be at least 70% (e.g., at

GenBank Accession Nos. NP_037501.2 and/or NP_787063.1.

A DNMT

[00249] A DNMT may may be inhibited be inhibited using using any any suitable suitable method method known known in the in the art.art. Suitable Suitable

methods include knockdown of a DNMT mRNA, genetically knocking out a DNMT, and use

of a DNMT inhibitor (e.g., chemical inhibitors). DNMT inhibitors are being investigated in

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

clinical trials (e.g., phase III clinical trials) in the United States of America and beyond. Non-

limiting examples of DNMT inhibitors include VIDAZATM (azacitidine) VIDAZA (azacitidine) (e.g., (e.g., for for the the

treatment of Myelodysplastic Syndromes and treatment of acute myeloid leukemia (AML)),

DACOGEN (decitabine) (e.g., for treatment of AML and treatment of Chronic myeloid

leukemia (CML)), and Guadecitabine (SGI-110) (e.g., for treatment of AML). In 2012, the

European Union approved DACOGEN (decitabine) for use in patients with AML.

[00250] A DNMT may be inhibited by inhibiting a DNMT stabilizer. Suitable methods of

inhibiting a DNMT stabilizer include knockdown of the mRNA encoding the stabilizer,

genetically knocking out the gene that encodes the stabilizer and use of an inhibitor (e.g.,

chemical inhibitors). As a non-limiting example, KDM1a, which is also referred to as Lsd1

or Aof2, is a stabilizer of DNMT1. See, e.g., Wang et al., Nat Genet. 2009 Jan;4 (1): 125-9. Jan;41(1):125-9.

In some embodiments, KDM1a KDM1: expression is knocked down using a shRNA disclosed herein

or known in the art. In some embodiments, KDM1a is inhibited to prevent injury induced by

hypermethylation from DNMTs, which could be useful in promoting reprogramming.

[00251] In some In some embodiments, embodiments, a histone a histone methyltransferase methyltransferase is aisreprogramming a reprogramming barrier barrier

and is inhibited to facilitate reprogramming of a cell, tissue and/or organ. Histone

methyltransferases may be inhibited by any suitable method, including use of chemical

inhibitors. For example, 3-deazaneplanocin A (Dznep), epz004777, and BIX-01294 are

examples of histone methyltransferase inhibitors.

[00252] In some In some embodiments, embodiments, a reprogramming a reprogramming barrier barrier is aishistone a histone deacetylase deacetylase (HDAC) (HDAC)

and a HDAC is inhibited to facilitate reprogramming of a cell, tissue, and/or organ. Non-

limiting examples of HDAC inhibitors include valproic acid (VPA), trichostatin A (TSA),

suberoylanilide hydroxamic Acid (SAHA), sodium butyrate (SB), Belinostat (PXD101),

Panobinostat (LBH589), Quisinostat (JNJ-26481585), Abexinostat (PCI-24781), Givinostat

(ITF2357), Resminostat (4SC-201), Phenylbutyrate (PBA), Depsipeptide (romidepsin),

Entinostat (MS-275), Mocetinostat (MGCD0103), and Tubastatin A (TBA).

[00253] In some embodiments, a reprogramming barrier is a NF-kB, and it is inhibited to

facilitate reprogramming of a cell, tissue, and/or organ. Non-limiting examples of NF-kB

inhibitor includes BAY 11-7082, TPCA 1, and p65 siRNA. See, e.g., the NF-kB small

molecule guide compiled by Abcam, which is available on the Abcam website

(www.abcam.com/reagents/nf-kb-small-molecule-guide). (www.abcam.com/reagents/nf-kb-small-molecule-guide)

[00254] In some embodiments, a repogramming barrier is a cytokine secreted from

senescent cells in which a cytokine is inhibited to facilitate reprogramming of a cell, tissue, and/or organ. None limiting examples of cytokines inhibitors include Anti-TNFa (Mahmoudi Anti-TNF (Mahmoudi et al, Biorxiv, 2018) and drugs, including Navitoclax, that kill senescence cells.

[00255] In In

[00255] someembodiments, some embodiments, a reprogramming reprogrammingbarrier is is barrier a microRNA (miRNA) a microRNA and a and a (miRNA)

microRNA is inhibited to facilitate reprogramming of a cell, tissue, and/or organ. Non-

limiting examples of microRNAs that are reprogramming barriers include miR Let-7 and

miR-34. Without being bound by a particular theory, inhibition of miR Let-7 may increase

the efficiency of reprogramming because miR Let-7 inhibits the cell cycle and inhibition of

miR-34 may facilitate reprogramming because miR-34 inhibits the translation of p53.

[00256] In In

[00256] someembodiments, some embodiments, OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,replacements thereof, replacements or any thereof, or any

combination thereof is activated in a cell, tissue, organ and/or a subject in combination with

inhibition of PTEN, SOCS3, RhoA, and/or ROCK to enhance nerve regeneration. In some

embodiments, PTEN is deleted, SOCS3 is deleted, RhoA is knocked down, and/or ROCK is

knocked down in a cell, tissue, organ and/or subject. See, e.g., Park et al., Science. 2008 Nov

7;322(5903):963-6; Smith et al., Neuron. 2009 Dec 10;64(5):617-23; Koch et al., Front Cell

Neurosci. 2014 Sep 5;8:273; Koch et al., Cell Death Dis. 2014 May 15;5:e1225 for

descriptions of inhibition of PTEN, SOCS3, RhoA, and/or ROCK. Each reference is hereby

incorporated by reference in its entirety for this purpose.

[00257]

[00257] In In someembodiments, some embodiments, OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,replacements thereof, replacements or any thereof, or any

neuronal electrical stimulation (e.g., high-contrast visual stimulation) to promote nerve

regeneration. regeneration. See, e.g., See, Lim Lim e.g., et al., Nat Neurosci. et al., 2016 Aug; Nat Neurosci. 19(8):1073-84 2016 for a description Aug;19(8):1073-84 for a description

of high-contrast visual stimulation. This reference is hereby incorporated by reference in its

entirety for this purpose.

[00258]

[00258] In In someembodiments, some embodiments, OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,replacements thereof, replacements or any thereof, or any

gamma band light stimulation to promote nerve regeneration. See, e.g., McDermott et al., J

Alzheimers Dis. 2018; 65(2): 363-392 for a description of gamma band light stimulation.

This reference is hereby incorporated by reference in its entirety for this purpose.

Engineered cells

[00259] Engineered cells and method of producing engineered cells are also encompassed

by the present disclosure. The engineered cells, for example, may be useful in cell-based

therapies (e.g., stem cell therapies). Although stem cell therapy is currently in clinical trials

(see, e.g., David Cyranoski, Nature 557, 619-620 (2018), toxicity (e.g., off-target toxicity) is

PCT/US2019/053545

a concern, Without being bound by a particular theory, the engineered cells of the present

disclosure (e.g., cells engineered using AAV vectors encoding OCT4, KLF4, and/or SOX2,

and/or an inducing agent) may have a lower toxicity because AAV is does not integrate into

the genome of host cells and use of the inducible systems described herein to control

expression of OCT4, KLF4, and/or SOX2 may allow for precise control (e.g., amount and

timing) of gene expression.

[00260] Any of the nucleic acids (e.g., engineered nucleic acid) capable of inducing

OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), engineered proteins

described herein, chemical agents activating (e.g., inducing expression of) OCT4, KLF4,

and/or SOX2, antibodies activating (e.g., inducing expression of) OCT4, KLF4, and/or

SOX2, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus,

retrovirus, herpes virus, or AAV) described herein, alone, or in combination may be

introduced into a host cell, host tissue, or organ to produce an engineered cell, an engineered

tissue, or an engineered organ. Any of the nucleic acids (e.g., engineered nucleic acid) (e.g.,

expression vector) capable of inducing expression of OCT4; KLF4; SOX2; or any

combination thereof, engineered proteins described herein, chemical agents activating (e.g.,

inducing expression of) OCT4; KLF4; SOX2; or any combination thereof, antibodies

activating (e.g., inducing expression of) OCT4; KLF4; SOX2; or any combination thereof,

and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus,

herpes virus, or AAV) described herein, alone, or in combination may be introduced into a

host cell, host tissue, or organ to produce an engineered cell, an engineered tissue, or an

engineered organ. In some embodiments, a nucleic acid (e.g., engineered nucleic acid)

encoding an inducing agent, an engineered protein encoding an inducing agent, a chemical

agent capable of modulating (e.g., activating or inhibiting) the activity of an inducing agent,

and/or a recombinant virus encoding an inducing agent is also introduced into a host cell, host

tissue, or organ to produce an engineered cell, an engineered tissue, or an engineered organ.

[00261] In some In some embodiments, embodiments, the the engineered engineered cellcell is induced is an an induced pluripotent pluripotent stemstem cellcell

(iPSC).

In some

[00262] In some embodiments, embodiments, a viral a viral vector vector (e.g., (e.g., an AAV an AAV vector, vector, including including a vector a vector

with a TRE promoter operably linked to a nucleic acid encoding OCT4, KLF4, and SOX2) is

packaged into a virus with an AAV-DJ capsid. In some embodiments, the AAV-DJ capsid

increases the transduction efficiency into cultured cells compared to cells without the AAV-

DJ capsid. In some embodiments, the AAV virus encoding OSK is administered to a cell. In

some embodiments, an AAV virus (e.g., AAV-DJ virus) encoding the inducing agent or a

WO wo 2020/069373 PCT/US2019/053545

protein encoding the inducing agent is administered to the same cells. In some embodiments,

this system produces an engineered cell (e.g., an induced pluripotent stem cell). In some

embodiments, the engineered cell is further differentiated into (e.g., differentiated into an eye,

ear, nose, mouth including gum and roots of teeth, bone, lung, breast, udder, pancreas,

stomach, oesophagus, muscle including cardiac muscle, liver, blood vessel, skin including

hair, heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine

cell). In some embodiments, the differentiated cell is used for transplantation purposes. In In

some embodiments, the engineered cell is cultured to create an engineered tissue. In some

embodiments, the engineered cell is cultured to create an engineered organ. In some

embodiments, the engineered cells are retina pigment epithelium cells, neuron cells,

pancreatic beta-cells, or cardiac cells.

Compositions

[00263] The The compositions compositions of the of the disclosure disclosure may may comprise comprise at least at least one one of any of any of the of the

nucleic acids (e.g., engineered nucleic acid) capable of inducing OCT4, KLF4, and/or SOX2

expression (e.g., expression vector), engineered proteins, engineered cells, chemical agents

activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2, antibodies activating

(e.g., inducing expression of) OCT4, KLF4, and/or SOX2, and/or recombinant viruses (e.g.,

herein alone, or in combination. In certain embodiments, the compositions of the disclosure

comprise at least one of any of the nucleic acids (e.g., engineered nucleic acid) (e.g.,

expression vector) capable of inducing expression of OCT4; KLF4; SOX2; or any

combination thereof, engineered proteins, engineered cells, chemical agents activating (e.g.,

and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus,

retrovirus, herpes virus, or AAV) described herein alone, or in combination. In some

embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different nucleic

acids (e.g., engineered nucleic acids) capable of inducing OCT4, KLF4, and/or SOX2

expression (e.g., expression vectors encoding OCT4, KLF4, and/or SOX2). In some

acids (e.g., engineered nucleic acids) capable of inducing expression of OCT4; KLF4; SOX2;

or any combination thereof (e.g., expression vectors encoding OCT4; KLF4; SOX2; or any

combination thereof). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,

WO wo 2020/069373 PCT/US2019/053545

10, or more different viruses (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus,

retrovirus, herpes virus, or AAV) each having one or more different transgenes. In some

embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different chemical

agents activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2. In some

agents activating (e.g., inducing expression of) OCT4; KLF4; SOX2; or any combination

thereof. In some embodiments, a composition further comprises one or more nucleic acids

(e.g., engineered nucleic acids) encoding an inducing agent, one or more engineered proteins

encoding an inducing agent, one or more chemical agents capable of modulating (e.g.,

activating or inhibiting) the activity of an inducing agent, and/or one or more recombinant

viruses encoding an inducing agent. In some embodiments, a composition comprises

engineered cells (e.g., induced pluripotent stem cells and/or differentiated cells). In some

embodiments, a composition comprises an engineered protein encoding OCT4, SOX2, and/or

KLF4. In some embodiments, a composition comprises an engineered protein encoding

OCT4, SOX2, KLF4, or any combination thereof. In some embodiments, a composition

further comprises an engineered protein encoding an inducing agent.

In some

[00264] In some embodiments, embodiments, a composition a composition further further comprises comprises a pharmaceutically a pharmaceutically

acceptable carrier. Suitable carriers may be readily selected by one of skill in the art in view

of the indication for which the nucleic acids (e.g., engineered nucleic acid) capable of

inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), chemical agents

(e.g., inducing expression of) OCT4, KLF4, and/or SOX2, engineered proteins, engineered

cells, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus,

retrovirus, herpes virus, or AAV) e.g. is directed. Suitable carriers may be readily selected by

one of skill in the art in view of the indication for which the nucleic acids (e.g., engineered

nucleic acid) (e.g., expression vectors) capable of inducing expression of OCT4; KLF4;

SOX2; or any combination thereof, chemical agents activating (e.g., inducing expression of)

OCT4; KLF4; SOX2; or any combination thereof, antibodies activating (e.g., inducing

expression of) OCT4; KLF4; SOX2; or any combination thereof, engineered proteins,

engineered cells, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia

virus, retrovirus, herpes virus, or AAV) e.g. is directed. Suitable carriers may also be readily

selected by one of skill in the art in view of the indication for which the nucleic acids (e.g.,

engineered nucleic acids) encoding an inducing agent, engineered proteins encoding an

inducing agent, chemical agents capable of modulating (e.g., activating or inhibiting) the

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

activity of an inducing agent, and/or recombinant viruses (e.g., lentivirus, adenovirus,

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) comprising an inducing agent

e.g. is directed. For example, one suitable carrier includes saline, which may be formulated

with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary

carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar,

pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the

present disclosure.

Optionally,

[00265] Optionally, the the compositions compositions of the of the disclosure disclosure may may comprise, comprise, in addition in addition to the to the

expression (e.g., expression vector), engineered cells comprising OCT4, KLF4, and/or SOX2,

engineered proteins, chemical agents activating (e.g., inducing expression of) OCT4, KLF4,

retrovirus, herpes virus, or AAV) e.g. and carrier(s), other pharmaceutical ingredients, such

as preservatives, or chemical stabilizers. Optionally, the compositions of the disclosure may

comprise, in addition to the nucleic acids (e.g., engineered nucleic acid) (e.g., expression

vector) capable of inducing expression of OCT4; KLF4; SOX2; or any combination thereof,

engineered cells comprising OCT4; KLF4; SOX2; or any combination thereof, engineered

proteins, chemical agents activating (e.g., inducing expression of) OCT4; KLF4; SOX2; or

any combination thereof, antibodies activating (e.g., inducing expression of) OCT4; KLF4;

SOX2; or any combination thereof, and/or recombinant viruses (e.g., lentivirus, adenovirus,

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) e.g. and carrier(s), other

pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary

preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl

gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable

chemical stabilizers include gelatin and albumin. The compositions of the present disclosure

may further comprise a nucleic acid (e.g., engineered nucleic acids) encoding an inducing

agent, an engineered protein encoding an inducing agent, chemical agents capable of

modulating (e.g., activating or inhibiting) the activity of an inducing agent, and/or

recombinant viruses encoding an inducing agent.

[00266] The The nucleic nucleic acid(e.g., acid (e.g., engineered engineered nucleic nucleicacid) (e.g., acid) expression (e.g., vector) expression capablecapable vector)

of inducing expression of OCT4; KLF4; SOX2; or any combination thereof, engineered cells,

combination thereof, engineered proteins encoding OCT4; KLF4; SOX2; or any combination

WO wo 2020/069373 PCT/US2019/053545

thereof, antibodies activating (e.g., inducing expression of) OCT4; KLF4; SOX2; or any

combination thereof, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

vaccinia virus, retrovirus, herpes virus, or AAV) encoding the same described herein are

administered in sufficient amounts to transfect the cells of a desired tissue (e.g., eye, ear,

nose, mouth including gum and roots of teeth, bone, lung, breast, udder, pancreas, stomach,

oesophagus, muscle including cardiac muscle, liver, blood vessel, skin including hair, heart,

brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine tissue) and to

provide sufficient levels of gene transfer and expression without undue adverse effects. Any

of the nucleic acids (e.g., engineered nucleic acids) encoding an inducing agent, an

engineered protein encoding an inducing agent, chemical agents capable of modulating (e.g.,

activating or inhibiting) the activity of an inducing agent, and/or recombinant viruses

encoding an inducing agent are administered in sufficient amounts to transfect the cells of a

desired tissue (e.g., eye, ear, nose, mouth including gum and roots of teeth, bone, lung, breast,

udder, pancreas, stomach, oesophagus, muscle including cardiac muscle, liver, blood vessel,

skin including hair, heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary,

or intestine tissue) and to provide sufficient levels of gene transfer and expression without

undue adverse effects. Examples of pharmaceutically acceptable routes of administration

include, but are not limited to, direct delivery to the selected organ (e.g., direct delivery to

eye, ear, nose, mouth including gum and roots of teeth, bone, lung, breast, udder, pancreas,

hair, heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine).

Any of the nucleic acids (e.g., engineered nucleic acids) capable of inducing OCT4, KLF4,

and/or SOX2 expression (e.g., expression vector), engineered cells, chemical agents

activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2, engineered proteins,

antibodies activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2, and/or

recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus,

herpes virus, or AAV) described herein may be delivered intravenously, intradermally,

WO wo 2020/069373 PCT/US2019/053545

skill in the art. Any of the nucleic acids (e.g., engineered nucleic acids) (e.g., expression

vectors) capable of inducing expression of OCT4; KLF4; SOX2; or any combination thereof,

engineered cells, chemical agents activating (e.g., inducing expression of) OCT4; KLF4;

SOX2; or any combination thereof, engineered proteins, antibodies activating (e.g., inducing

expression of) OCT4; KLF4; SOX2; or any combination thereof, and/or recombinant viruses

described herein may be delivered intravenously, intradermally, intraarterially,

intralesionally, intratumorally, intracranially, intraarticularly, intraprostaticaly, intrapleurally,

intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally,

intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,

mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally,

systemically, injection, infusion, continuous infusion, localized perfusion bathing target cells

directly, via a catheter, in creams, in lipid compositions (e.g., liposomes), or by other method

or any combination of the forgoing as would be known to one of ordinary skill in the art.

Any of the nucleic acids encoding an inducing agent, chemical agents capable of modulating

the activity of an inducing agent, engineered proteins encoding an inducing agent, and/or

recombinant viruses encoding an inducing agent may be may be delivered intravenously,

intraprostaticaly, intraprostaticaly, intrapleurally, intrapleurally, intranasally, intranasally, intravitreally, intravitreally, intravaginally, intravaginally, intrarectally, intrarectally,

skill in the art. Routes of administration may be combined, if desired.

In some

[00267] In some embodiments,a a nucleic embodiments, nucleic acid acid is isdelivered deliverednon-virally (e.g., non-virally not onnot (e.g., a viral on a viral

vector and/or not in a virus). In some embodiments, a nucleic acid (e.g., RNA or DNA)

encoding OCT4, SOX2, and/or KLF4 and/or an inducing agent is administered in a liposome.

In some embodiments, a nucleic acid (e.g., RNA or DNA) encoding OCT4, SOX2, KLF4, or

any combination thereof, and/or an inducing agent is administered in a liposome. In some

embodiments, a nucleic acid (e.g., RNA or DNA) encoding OCT4, SOX2, and/or KLF4

and/or an inducing agent is administered in a particle. In some embodiments, a nucleic acid

(e.g., RNA or DNA) encoding OCT4, SOX2, KLF4, or any combination thereof, and/or an

WO wo 2020/069373 PCT/US2019/053545

inducing agent is administered in a particle. In some embodiments, the nucleic acid is RNA

(e.g., mRNA).

In some

[00268] In some embodiments, embodiments, a pharmaceutical a pharmaceutical composition composition comprising comprising an expression an expression

vector encoding OCT4, KLF4, and/or SOX2 or a pharmaceutical composition comprising a

virus harboring the expression vector is administered to a cell, tissue, organ or a subject. In

some embodiments, a pharmaceutical composition comprising an expression vector encoding

an inducing agent or a pharmaceutical composition comprising a virus harboring the

expression vector is administered to a cell, tissue, organ or a subject. In some embodiments,

the virus and/or expression vector encoding OCT4, KLF4, and/or SOX2 is administered

systemically. In some embodiments, the virus and/or expression vector encoding an inducing

agent is administered systemically. In some embodiments, the virus and/or expression vector

encoding OCT4, KLF4, and/or SOX2 is administered locally (e.g., directly to a tissue or

organ of interest, including eye, ear, nose, mouth including gum and roots of teeth, bone,

lung, breast, udder, pancreas, stomach, oesophagus, muscle including cardiac muscle, liver,

blood vessel, skin including hair, heart, brain, nerve tissue, kidney, testis, prostate, penis,

cloaca, fin, ovary, or intestine). In some embodiments, a virus and/or expression vector

encoding an inducing agent is administered locally (e.g., directly to a tissue or organ of

interest, including eye, ear, nose, mouth including gum and roots of teeth, bone, lung, breast,

or intestine). In some embodiments, the inducing agent (e.g., a nucleic acid encoding the

inducing agent, a protein encoding the inducing agent, or a virus encoding the inducing

agent) and/or chemical agent capable of modulating (e.g., activating or inhibiting) the activity

of the inducing agent is administered using the same route of administration as the OCT4,

KLF4, and/or SOX2 (e.g., nucleic acid encoding OCT4, KLF4, and/or SOX2). In some

embodiments, the inducing agent (e.g., a nucleic acid encoding the inducing agent, a protein

encoding the inducing agent, or a virus encoding the inducing agent) and/or chemical agent

capable of modulating (e.g., activating or inhibiting) the activity of the inducing agent is

administered via a different route of administration as the OCT4, KLF4, and/or SOX2 (e.g.,

nucleic acid encoding OCT4, KLF4, and/or SOX2).

[00269] In some In some embodiments, embodiments, a pharmaceutical a pharmaceutical composition composition comprising comprising an expression an expression

vector encoding OCT4; KLF4; SOX2; or any combination thereof, or a pharmaceutical

composition comprising a virus harboring the expression vector is administered to a cell,

tissue, organ, or subject. In some embodiments, a pharmaceutical composition comprising an

WO wo 2020/069373 PCT/US2019/053545

expression vector encoding an inducing agent or a pharmaceutical composition comprising a

virus harboring the expression vector is administered to a cell, tissue, organ, or subject. In

some embodiments, the virus and/or expression vector encoding OCT4; KLF4; SOX2; or any

combination thereof is administered systemically. In some embodiments, the virus and/or

expression vector encoding an inducing agent is administered systemically. In some

embodiments, the virus and/or expression vector encoding OCT4; KLF4; SOX2; or any

combination thereof is administered locally (e.g., directly to a tissue or organ of interest,

including eye, ear, nose, mouth including gum and roots of teeth, bone, lung, breast, udder,

intestine). In some embodiments, a virus and/or expression vector encoding an inducing

agent is administered locally (e.g., directly to a tissue or organ of interest, including eye, ear,

brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine). In some

administered using the same route of administration as the OCT4; KLF4; SOX2; or any

combination thereof (e.g., nucleic acid encoding OCT4; KLF4; SOX2; OCT4 and SOX2;

OCT4 and KLF4; KLF4 and SOX2; or KLF4, OCT4, and SOX2). In some embodiments, the

inducing agent (e.g., a nucleic acid encoding the inducing agent, a protein encoding the

inducing agent, or a virus encoding the inducing agent) and/or chemical agent capable of

modulating (e.g., activating or inhibiting) the activity of the inducing agent is administered

via a different route of administration as the OCT4; KLF4; SOX2; or any combination

thereof (e.g., nucleic acid encoding nucleic acid encoding OCT4; KLF4; SOX2; OCT4 and

SOX2; OCT4 and KLF4; KLF4 and SOX2; or KLF4, OCT4, and SOX2).

[00270] In some embodiments, the expression vector is an inducible vector in which a

nucleic acid encoding OCT4, KLF4, and/or SOX2 and/or inducing agent, is operably linked

to an inducible TRE promoter (e.g., TRE3G, TRE2, or P tight). In some embodiments, the

expression vector is an inducible vector in which a nucleic acid encoding OCT4; KLF4;

SOX2; or any combination thereof, and/or inducing agent, is operably linked to an inducible

TRE promoter (e.g., TRE3G, TRE2, or P tight). In some embodiments, the virus and/or

inducible vector is administered with tetracycline (e.g., doxycycline). In some embodiments,

WO wo 2020/069373 PCT/US2019/053545

the virus and/or expression vector comprising a TRE promoter is administered separately

from tetracycline (e.g., doxycycline). For example, any of the viruses and/or expression

vectors comprising a TRE promoter described herein may be administered systemically and

the tetracycline may be administered locally (e.g., to an organ or tissue of interest). In some

embodiments, any of the viruses and/or expression vectors comprising a TRE promoter

described herein may be administered locally (e.g., to directly to a tissue or organ of interest,

intestine) and the tetracycline may be administered systemically. As a non-limiting example,

a virus and/or expression vector comprising a TRE promoter is administered directly (e.g.,

injected) into the eye of a subject and the tetracycline (e.g., doxycycline) is administered

systemically (e.g., orally as a pill).

[00271] In some In some embodiments, embodiments, tetracycline tetracycline is administered is administered intravenously, intravenously, intradermally, intradermally,

bathing target cells directly, via a catheter, in creams, or in lipid compositions. In some

embodiments, tetracycline is administered directly to a cell, organ, and/or tissue. As a non-

limiting example, tetracycline may be administered to the eye of a subject through any

suitable method, including eye drops comprising tetracycline, sustained release devices (e.g.,

micropumps, particles, and/or drug depots), and medicated contact lenses comprising

tetracycline. In some embodiments, tetracycline is administered systemically (e.g., through

drinking water or intravenous injection) to a subject. Tetracycline may be administered

topically (e.g., in a cream) or through a subcutaneous pump (e.g., to deliver tetracycline to a

particular tissue).

As example,

[00272] As an an example, thedose the dose of of recombinant recombinant virus virus(e.g., lentivirus, (e.g., alphaviruses, lentivirus, alphaviruses,

vaccinia viruses, adenovirus, retrovirus, herpes virus, or AAV) virions required to achieve a

particular therapeutic effect, e.g., the units of dose in genome copies/per kilogram of body

weight (GC/kg), will vary based on several factors including, but not limited to: the route of

recombinant virus (e.g., lentivirus, alphaviruses, vaccinia viruses, adenovirus, retrovirus,

herpes virus, or AAV) virion administration, the level of gene or RNA expression required to

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of

the gene or RNA product. One of skill in the art can readily determine a recombinant virus

(e.g., lentivirus, alphaviruses, vaccinia viruses, adenovirus, retrovirus, herpes virus, or AAV

virion) dose range to treat a patient having a particular disease or disorder based on the

aforementioned factors, as well as other factors.

An effective

[00273] An effective amountof amount ofaa recombinant recombinant virus virus(e.g., lentivirus, (e.g., alphaviruses, lentivirus, alphaviruses,

vaccinia viruses, adenovirus, retrovirus, herpes virus, or AAV) is an amount sufficient to

target infect an animal, target a desired tissue. In some embodiments, an effective amount of

an recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus,

herpes virus, or AAV) is an amount sufficient to produce a stable somatic transgenic animal

model. The effective amount will depend primarily on factors such as the species, age,

weight, health of the subject, and the tissue to be targeted, and may thus vary among animal

and tissue. For example, an effective amount of the recombinant virus (e.g., lentivirus,

alphavirus, vaccinia virus, adenovirus, retrovirus, herpes virus, or AAV) is generally in the

range range of offrom fromabout 1 ml about 1 to ml about 100 ml to about of ml 100 solution containing of solution from aboutfrom containing 109 about to 10 16 10 to 10¹

genome copies. In some cases, a dosage between about 1011 10¹¹ to 1013 10¹³ recombinant virus (e.g.,

lentivirus, adenovirus, retrovirus, alphavirus, vaccinia virus, herpes virus, or AAV) genome

copies is appropriate. In certain embodiments, 1010 or 10¹¹ 10¹ or 1011 recombinant recombinant virus virus (e.g., (e.g., lentivirus, lentivirus,

adenovirus, retrovirus, alphavirus, vaccinia virus, herpes virus, or AAV) genome copies is

effective to target ocular tissue (e.g., retinal tissue). In some cases, stable transgenic animals

are produced by multiple doses of a recombinant virus (e.g., lentivirus, adenovirus, retrovirus,

herpes virus, alphavirus, vaccinia virus, or AAV).

In some

[00274] In some embodiments, embodiments, a dose a dose of recombinant of recombinant virus virus (e.g., (e.g., lentivirus, lentivirus, adenovirus, adenovirus,

retrovirus, herpes virus, alphavirus, vaccinia virus, or AAV) is administered to a subject no

more than once per calendar day (e.g., a 24-hour period). In some embodiments, a dose of

recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpes

virus, or AAV) is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar

days. In some embodiments, a dose of recombinant virus (e.g., lentivirus, alphavirus,

vaccinia virus, adenovirus, retrovirus, herpes virus, or AAV) is administered to a subject no

more than once per calendar week (e.g., 7 calendar days). In some embodiments, a dose of

virus, or AAV) is administered to a subject no more than bi-weekly (e.g., once in a two

calendar week period). In some embodiments, a dose of recombinant virus (e.g., lentivirus,

alphavirus, vaccinia virus, adenovirus, retrovirus, herpes virus, or AAV) is administered to a

WO wo 2020/069373 PCT/US2019/053545

subject no more than once per calendar month (e.g., once in 30 calendar days). In some

embodiments, a dose of recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus,

adenovirus, retrovirus, herpes virus, or AAV) is administered to a subject no more than once

per six calendar months. In some embodiments, a dose of recombinant virus (e.g., lentivirus,

subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

In some

[00275] In some embodiments, embodiments, recombinant recombinant virus virus (e.g., (e.g., lentivirus, lentivirus, alphavirus, alphavirus, vaccinia vaccinia

virus, adenovirus, retrovirus, herpes virus, or AAV) compositions are formulated to reduce

aggregation of AAV particles in the composition, particularly where high recombinant virus

(e.g., lentivirus, alphavirus, vaccinia virus, adenovirus, retrovirus, herpes virus, or AAV)

concentrations are present (e.g., ~10¹³ ~1013 GC/ml or more). Appropriate methods for reducing

aggregation of may be used, including, for example, addition of surfactants, pH adjustment,

salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005)

12, 171-178, the contents of which are incorporated herein by reference.)

[00276] As aAsnon-limiting a non-limiting example, example, delivery delivery of transgenes of transgenes via via AAV AAV havehave beenbeen shown shown to to

be feasible and non-toxic in humans. For example, AAV may be delivered to the eye. See,

e.g., Smalley Nat Biotechnol. 2017 Nov 9;35(11):998-999.

Formulation

[00277] Formulation of pharmaceutically-acceptable of pharmaceutically-acceptable excipients excipients and and carrier carrier solutions solutions is is

well-known to those of skill in the art, as is the development of suitable dosing and treatment

regimens for using the particular compositions described herein in a variety of treatment

regimens. Typically, these formulations may contain at least about 0.1% of the active

compound or more, although the percentage of the active ingredient(s) may, of course, be

varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the

weight or volume of the total formulation. Naturally, the amount of active compound in each

therapeutically-useful composition may be prepared is such a way that a suitable dosage will

be obtained be obtainedininanyany given unitunit given dose dose of the ofcompound. Factors Factors the compound. such as solubility, such as solubility,

bioavailability, biological half-life, route of administration, product shelf life, as well as other

pharmacological considerations will be contemplated by one skilled in the art of preparing

such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens

may be desirable.

[00278] In some In some embodiments, embodiments, the the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acid) acid) capable capable of of

inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), engineered cells

comprising OCT4, KLF4, and/or SOX2, engineered proteins encoding Oct4, KLF4, and/or

SOX2, chemical agents activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2,

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

herpes virus, or AAV) e.g. in suitably formulated pharmaceutical compositions disclosed

herein are delivered directly to target tissue, e.g., direct to a tissue of interest (e.g., eye, ear,

brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine).

In some

[00279] In some embodiments,the embodiments, the nucleic nucleic acids acids(e.g., (e.g.,engineered nucleic engineered acid) acid) nucleic (e.g.,(e.g.,

expression vector) capable of inducing expression of OCT4; KLF4; SOX2; or any

combination thereof, engineered cells comprising OCT4; KLF4; SOX2; or any combination

thereof, engineered proteins encoding Oct4, KLF4, SOX2, or a combination thereof,

chemical agents activating (e.g., inducing expression of) OCT4, KLF4, SOX2, or a

combination thereof, antibodies activating (e.g., inducing expression of) OCT4, KLF4,

SOX2, or a combination thereof, and/or recombinant viruses (e.g., lentivirus, adenovirus,

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) e.g. in suitably formulated

pharmaceutical compositions disclosed herein are delivered directly to target tissue, e.g.,

direct to a tissue of interest (e.g., eye, ear, nose, mouth including gum and roots of teeth,

bone, lung, breast, udder, pancreas, stomach, oesophagus, muscle including cardiac muscle,

liver, blood vessel, skin including hair, heart, brain, nerve tissue, kidney, testis, prostate,

penis, cloaca, fin, ovary, or intestine).

[00280] In some In some embodiments, embodiments, the the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acid) acid) encoding encoding

an inducing agent (e.g., an expression vector), engineered cells comprising an inducing agent,

engineered proteins encoding a inducing agent, chemical agents capable of modulating the

activity of an inducing agent, and/or recombinant viruses (e.g., lentiviruses, adenoviruses,

alphaviruses, vaccinia viruses, retroviruses, herpes viruses, or AAVs) encoding an inducing

agent e.g. in suitably formulated pharmaceutical compositions disclosed herein are delivered

directly to target tissue, e.g., direct to a tissue of interest (e.g., eye, ear, nose, mouth including

gum and roots of teeth, bone, lung, breast, udder, pancreas, stomach, oesophagus, muscle

including cardiac muscle, liver, blood vessel, skin including hair, heart, brain, nerve tissue,

kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine).

However,

[00281] However, in in certaincircumstances certain circumstances it itmay maybebe desirable to separately desirable or in or to separately addition in addition

deliver any of the nucleic acids (e.g., engineered nucleic acid) capable of inducing OCT4,

KLF4, and/or SOX2 expression (e.g., expression vector) and/or nucleic acid encoding an

inducing agent, nucleic acids (e.g., engineered nucleic acid) capable of inducing expression

WO wo 2020/069373 PCT/US2019/053545

of a combination of transcription factors selected from OCT4, KLF4, and/or nucleic acid

encoding an inducing agent, engineered cells, engineered proteins, chemical agents activating

(e.g., inducing expression of) OCT4, KLF4, and/or SOX2, chemical agents activating (e.g.,

inducing expression of) a combination of transcription factors selected from OCT4, KLF4,

and SOX2, chemical agents capable of modulating (e.g., inhibiting or activating) the activity

of an inducing agent, antibodies activating (e.g., inducing expression of) OCT4, KLF4,

and/or SOX2, antibodies activating (e.g., inducing expression of) OCT4; KLF4; SOX2; or

any combination thereof, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

vaccinia virus, retrovirus, herpes virus, or AAV) via another route, e.g., subcutaneously,

intraopancreatically, intranasally, parenterally, intravenously, intramuscularly, intrathecally,

or orally, intraperitoneally, or by inhalation. In some embodiments, the administration

modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each

specifically incorporated herein by reference in its entirety) may be used to deliver

virus, or AAVs). In some embodiments, a preferred mode of administration is by

intrastromal injection.

[00282] In In

[00282] someembodiments, some embodiments, a nucleic nucleicacid acid(e.g., mRNA) (e.g., encoding mRNA) OCT4,OCT4, encoding SOX2, SOX2,

KLF4, or any combination thereof is nanoformulated into a polyplex, which may be useful,

for example, for noninvasive aerosol inhalation and delivery of the nucleic acid to the lung

(e.g., lung epithelium). See, e.g., Patel et al., Adv Mater. 2019 Jan 4:e1805116. doi:

10.1002/adma.201805116 for description of nanoformulated mRNA polyplexes, which is

hereby incorporated by reference in its entirety for this purpose.

[00283] The The pharmaceutical pharmaceutical forms forms suitable suitable for for injectable injectable use use include include sterile sterile aqueous aqueous

solutions or dispersions and sterile powders for the extemporaneous preparation of sterile

injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid

polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage

and use, these preparations contain a preservative to prevent the growth of microorganisms.

In many cases the form is sterile and fluid to the extent that easy syringability exists. It must

be stable under the conditions of manufacture and storage and must be preserved against the

contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a

solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol,

propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof,

and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a

coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00284] For For administrationofofan administration an injectable injectable aqueous aqueoussolution, for for solution, example, the solution example, the solution

may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with

sufficient saline or glucose. These particular aqueous solutions are especially suitable for

intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this

connection, a suitable sterile aqueous medium may be employed. For example, one dosage

may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of

hypodermoclysis fluid or injected at the proposed site of infusion, (see for example,

"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580).

Some variation in dosage will necessarily occur depending on the condition of the host. The

person responsible for administration will, in any event, determine the appropriate dose for

the individual host.

[00285] Sterile injectable solutions are prepared by incorporating the nucleic acid (e.g.,

expression vector), engineered cells, engineered proteins, chemical agents activating (e.g.,

inducing expression of) OCT4, KLF4, an and/or SOX2, antibodies activating (e.g., inducing

expression of) OCT4, KLF4, and/or SOX2, and/or active recombinant viruses (e.g.,

lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) e.g. in the

required amount in the appropriate solvent with various of the other ingredients enumerated

herein, as required, followed by filtered sterilization. Sterile injectable solutions are prepared

by incorporating the nucleic acid (e.g., engineered nucleic acid) (e.g., expression vector)

capable of inducing expression of OCT4, KLF4, SOX2 or any , or combination any thereof, combination thereof,

engineered cells, engineered proteins, chemical agents activating (e.g., inducing expression

of) OCT4; KLF4; SOX2; or any combination thereof, antibodies activating (e.g., inducing

expression of) OCT4; KLF4; SOX2; or any combination thereof, and/or active recombinant

AAV) e.g. in the required amount in the appropriate solvent with various of the other

ingredients enumerated herein, as required, followed by filtered sterilization. In certain

embodiments, the sterile injectable solutions are prepared by incorporating a nucleic acid

108

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

(e.g., engineered nucleic acid) encoding an inducing agent, engineered protein encoding an

inducing agent, chemical agents capable of modulating the activity of an inducing agent

and/or active recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus,

retrovirus, herpes virus, or AAV) encoding an inducing agent e.g. in the required amount in

the appropriate solvent with various of the other ingredients enumerated herein, as required,

followed by filtered sterilization. Generally, dispersions are prepared by incorporating the

various sterilized active ingredients into a sterile vehicle which contains the basic dispersion

medium and the required other ingredients from those enumerated above. In the case of

sterile powders for the preparation of sterile injectable solutions, the preferred methods of

preparation are vacuum-drying and freeze-drying techniques which yield a powder of the

active ingredient plus any additional desired ingredient from a previously sterile-filtered

solution thereof.

[00286] The The compositions compositions comprising comprising nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

encoding OCT4, KLF4, and/or SOX2 (e.g., expression vector), engineered cells, engineered

proteins, chemical agents activating (e.g., inducing expression of) OCT4, KLF4, and/or

SOX2, antibodies activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2,

retrovirus, herpes virus, or AAV) disclosed herein may also be formulated in a neutral or salt

form. The compositions comprising nucleic acids (e.g., engineered nucleic acids) (e.g.,

expression vector) encoding OCT4; KLF4; SOX2; or any combination thereof, engineered

cells, engineered proteins, chemical agents activating (e.g., inducing expression of) OCT4;

KLF4; SOX2; or any combination thereof, antibodies activating (e.g., inducing expression of)

OCT4; KLF4; SOX2; or any combination thereof, and/or recombinant viruses (e.g.,

lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) disclosed

herein may also be formulated in a neutral or salt form. The compositions may comprise an

inducing agent (e.g., a nucleic acid encoding an inducing agent or a protein encoding an

inducing agent and/or a recombinant virus encoding an inducing agent) and/or a chemical

agent capable of modulating the activity of an inducing agent. Pharmaceutically-acceptable

salts, include the acid addition salts (formed with the free amino groups of the protein) and

which are formed with inorganic acids such as, for example, hydrochloric or phosphoric

acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed

with the free carboxyl groups can also be derived from inorganic bases such as, for example,

sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as

isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions

109 will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

A carrier

[00287] A carrier includesany includes any and and all all solvents, solvents,dispersion media, dispersion vehicles, media, coatings, vehicles, coatings,

diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers,

carrier solutions, suspensions, colloids, and the like. The use of such media and agents for

pharmaceutical active substances is well known in the art. Supplementary active ingredients

can also be incorporated into the compositions.

[00288] Delivery Delivery vehicles vehicles suchsuch as liposomes, as liposomes, nanocapsules, nanocapsules, microparticles, microparticles, microspheres, microspheres,

lipid particles, vesicles, and the like, may be used for the introduction of the compositions of

the present disclosure into suitable host cells. In particular, any of the nucleic acids (e.g.,

engineered nucleic acids) capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g.,

expression vector), any of the engineered proteins, any of the chemical agents activating (e.g.,

inducing expression of) OCT4, KLF4, and/or SOX2, any of the antibodies activating (e.g.,

inducing expression of) OCT4, KLF4, and/or SOX2, engineered cells, and/or any of the

herpes virus, or AAV) may be encapsulated in a lipid particle, a liposome, a vesicle, a

nanosphere, or a nanoparticle or the like. In some embodiments, any of the nucleic acids

(e.g., engineered nucleic acids) (e.g., expression vector) capable of inducing expression of

OCT4; KLF4; SOX2; or any combination thereof, any of the engineered proteins, any of the

combination thereof, any of the antibodies activating (e.g., inducing expression of) OCT4;

KLF4; SOX2; or any combination thereof, engineered cells, and/or any of the recombinant

AAV) may be encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a

nanoparticle or the like. An inducing agent (e.g., a nucleic acid encoding an inducing agent

or a protein encoding an inducing agent and/or a recombinant virus encoding an inducing

agent) and/or a chemical agent capable of modulating the activity of an inducing agent may

be encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or

the the like. like.

[00289] In some In some embodiments, embodiments, the the delivery delivery vehicle vehicle targets targets the the cargo. cargo. For For example, example, any any of of

the nucleic acids, engineered proteins, chemical agents, antibodies, and/or recombinant

AAV) described herein may be delivered via a nanoparticle that delivers the cargo to a

110

WO wo 2020/069373 PCT/US2019/053545

certain tissue or cell type. Nanoparticles coated in galactose polymers, for example, are

known to release their cargo within senescent cells as a result of their endogenous beta-

galactosidase activity. See e.g., Lozano-Torres et al., J Am Chem Soc. 2017 Jul

5;139(26):8808-8811. 5;139(26):8808-8811.

In some

[00290] In some embodiments, embodiments, any any of the of the nucleic nucleic acids, acids, engineered engineered proteins, proteins, chemical chemical

agents, antibodies, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

vaccinia virus, retrovirus, herpes virus, or AAV) is formulated in a poly(glycoamidoamine)

brush nanoparticles. See, e.g., Dong et al., Nano Lett. 2016 Feb 10;16(2):842-8.

[00291] In some In some embodiments, embodiments, any any of the of the nucleic nucleic acids, acids, engineered engineered proteins, proteins, chemical chemical

vaccinia virus, retrovirus, herpes virus, or AAV) is formulated in a lipid nanoparticle. See,

e.g., Cullis and Hope Mol Ther. 2017 Jul 5;25(7):1467-1475 5;25(7):1467-1475.In Insome someembodiments, embodiments,the thelipid lipid

nanoparticle comprises one or more membrane fusion proteins, which deliver plasmids

directly into the cytoplasm or the factors OCT4; KLF4; SOX2; or any combination thereof

may be fused directly to the targeting protein with or without nanoparticle encapsulation. In

some embodiments, the lipid nanoparticle is a Fusogenix lipid nanoparticle. In some

embodiments, the lipid nanoparticle is a "Wrapped Liposomes" (WL). See, e.g., Yamauchi

et al., Biochim Biophys Acta. 2006 Jan; 1758(1):90-7. In Jan;1758(1):90-7. In some some embodiments, embodiments, the the lipid lipid

nanoparticle is a PEGylated liposome (e.g., DOXIL M (e.g., (e.g., Allen Allen & & Hansen, Hansen, Biochim Biochim

Biophys Acta. 1991 Jul 1;1066(1):29-36.), 1, 2-dioleoyl-sn- glycerol-3

phosphatidylethanolamine (DOPE), a neutral helper lipid phosphatidylethanolamine (PE), or

combinations thereof (e.g., Farhood et al., Biochim Biophys Acta. 1995 May 4;1235(2):289-

95; Zhou & Huang, Biochim Biophys Acta. 1994 Jan 19;1189(2):195-203.). In some

embodiments, the lipid nanoparticle or fusion protein comprises employs a molecule or

protein to mimic methods employed by viruses for intracellular delivery of macromolecules

(e.g., Kobayashi et al., Bioconjug Chem. 2009 May 20;20(5):953-9), e.g., using a variety of

pH sensitive peptides such as vesicular stomatitis virus proteins (VSV G), phage coat proteins

and/or shGALA, and/or Fusion associated small transmembrane (FAST) proteins, e.g., avian

reovirus (ARV), nelson bay reovirus (NBV), and baboon reovirus (BBV), aquareovirus

reovirus (AQV) and reptilian reovirus (RRV), and/or Bombesin targeting peptide. See, e.g.,

Peisajovich et al., Eur J Biochem. 2002 Sep;269(17):4342-50.; Sakurai et al., 2011. See also

Nesbitt, Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with

Multifunctional FAST proteins, Western University Thesis, 2012.

https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=14&ved=2ahUKEwi https://www.google.com/url?sa=t&rct=j&4=&esrc=s&source=web&cd=14&ved=2ahUKEwi

WO wo 2020/069373 PCT/US2019/053545

X-

YW5puzfAhXGTd8KHUmCAT0QFjANegQIAhAB&url=http%3A%2F%2Fir.lib.uwo.ca%2 YW5puzfAhXGTd8KHUmCATOQFjANegQIAhAB&url=htp%3A2F2Fir.lib.uwo.ca2 Fcgi%2Fviewcontent.cgi%3Farticle%3D1571%26context%3Detd&usg=AOvVaw3A20aC Fcgi%2Fviewcontent.cgi%3Farticle%3D1571%26context%3DetdQusg=AOvVaw3A20aOef

HfJIJSZRR_-kPD HfJIJSZRR_kPD

[00292] In some embodiments, In some a nucleic embodiments, acidacid a nucleic (e.g., RNA RNA (e.g., or DNA, including or DNA, a plasmid) including a plasmid)

encoding OCT4, KLF4, SOX2, or a combination thereof is encapsulated in a Fusogenix lipid

nanoparticle. In some embodiments, a nucleic acid encoding an inducing agent (e.g., rtTA or

tTA) is encapsulated in a Fusogenix lipid nanoparticle. In some embodiments, a lipid

nanoparticle comprises a viral membrane protein. Without being bound by a particular

theory, a lipid nanoparticle may be non-toxic because it comprises a membrane fusion protein

that is not a viral membrane fusion protein. Non-limiting examples of membrane fusion

proteins include membrane fusion proteins disclosed in U.S. Patent No. 7,851,595, U.S.

Patent No. 8,252,901, International Application Publication No. WO 2012/040825, and

International Application Publication No. WO 2002/044206.

In some

[00293] In some embodiments, embodiments, a composition a composition of the of the present present disclosure disclosure (e.g., (e.g., comprising comprising a a

nucleic acid encoding OCT4, KLF4, SOX2, or a combination thereof) is delivered non-

virally. Methods of non-viral delivery of nucleic acids include lipofection, nucleofection,

microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or

lipid:nucleic acid conjugates, naked nucleic acid (e.g., RNA or DNA), artificial virions, and

agent-enhanced uptake of a nucleic acid (e.g., RNA or DNA).

In some

[00294] In some embodiments,a a cationic embodiments, cationic lipid lipidisisused to to used deliver a nucleic deliver acid. acid. a nucleic A A

cationic lipid is a lipid which has a cationic, or positive, charge at physiologic pH. Cationic

lipids can take a variety of forms including, but not limited to, liposomes or micelles.

Cationic lipids useful for certain aspects of the present disclosure are known in the art, and,

generally comprise both polar and non-polar domains, bind to polyanions, such as nucleic

acid molecules or negatively supercharged proteins, and are typically known to facilitate the

delivery of nucleic acids into cells. Examples of useful cationic lipids include

polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a

combination of DOTMA and DOPE, see, e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and

4,897,355), Lipofectase, LIPOFECTAMINER (e.g.,LIPOFECTAMINE® LIPOFECTAMINE (e.g., LIPOFECTAMINER2000, 2000,

LIPOFECTAMINER 3000, LIPOFECTAMINE© LIPOFECTAMINE® LIPOFECTAMINER RNAiMAX, LIPOFECTAMINE® LIPOFECTAMINER LTX), SAINT-RED (Synvolux Therapeutics, Groningen Netherlands), DOPE, Cytofectin (Gilead

Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary

cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-

-(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium chloride (DOTMA), N-[1 1 -(2,3-dioleoloxy)-propyl]-N,N,N-

trimethylammonium methylsulfate trimethylammonium (DOTAP), methylsulfate 3B-[N-(N',N'- (DOTAP), 3-[N-(N',N'-

limethylaminoethane)carbamoyl]cholesterol (DC-Chol), dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N- 2,3,-dioleyloxy-N-

2(sperminecarboxamido)ethy1]-N,N-dimethyl-1-propanaminiumt trifluoroacetate

[2(sperminecarboxamido)ethyl]-N,N-dimethy1-1-propanaminium trifluoroacetate (DOSPA), (DOSPA),

1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; bromide; and and

dimethyldioctadecylammonium bromide (DDAB). Cationic lipids have been used in the art

to deliver nucleic acid molecules to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548;

5,830,430; 5,780,053; 5,767,099; 8,569,256; 8,691,750; 8,748,667; 8,758,810; 8,759,104;

8,771,728; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998.

Molecular Membrane Biology 15:1).

[00295] In addition, other lipid compositions are also known in the art and include, e.g.,

those taught in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028;

U.S. Pat. No. 4,737,323. Cationic and neutral lipids that are suitable for efficient receptor-

recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO

91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues

(e.g. in vivo administration).

[00296] The preparation of lipid:nucleic acid complexes, including targeted liposomes

such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal,

Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al.,

Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994);

Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992);

U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,

4,837,028, and 4,946,787).

[00297] Polymer-based Polymer-based delivery delivery systems systems may may alsoalso be used be used to deliver to deliver a nucleic a nucleic acid. acid.

Polymers including polyethylenimine (PEI), chitosan, Poly (DL-Lactide) (DL- Lactide)(PLA) (PLA)and andPoly Poly((

DL-Lactide- co- glycoside) (PLGA), dedrimers, and Polymethacrylate may be used. See,

e.g., Yang et al., Macromol Biosci. 2012 Dec;12(12):1600-14; Ramamoorth et al., J Clin

Diagn Res. 2015 Jan; 9(1): GE01-GE06. As a non-limiting example, a cationic polymer may

be used. A cationic polymer is a polymer having a net positive charge. Cationic polymers

are well known in the art, and include those described in Samal et al., Cationic polymers and

their therapeutic potential. Chem Soc Rev. 2012 Nov 7;41(21):7147-94; in published U.S.

patent applications U.S. 2014/0141487 A1, U.S. 2014/0141094 A1, U.S. 2014/0044793 A1,

U.S. 2014/0018404 A1, U.S. 2014/0005269 A1, and U.S. 2013/0344117 A1; and in U.S. Pat.

Nos. 8,709,466; 8,728,526; 8,759,103; and 8,790,664; the entire contents of each are

113 incorporated herein by reference. Exemplary cationic polymers include, but are not limited to, polyallylamine (PAH); polyethyleneimine (PEI); poly(L-lysine) (PLL); poly(L-arginine)

(PLA); polyvinylamine homo- or copolymer; a poly(vinylbenzyl-tri-C1-C4-alkylammonium

salt); a polymer of an aliphatic or araliphatic dihalide and an aliphatic N,N,N',N'-tetra-C1-C4- N,N,N",N'-tetra-C1-C4-

alkyl-alkylenediamine; a poly(vinylpyridin) or poly(vinylpyridinium salt); a poly(N,N-

diallyl-N,N-di-C1-C4-alkyl-ammoniumhalide) aa homo- diallyl-N,N-di-C1-C4-alkyl-ammoniumhalide). homo- or or copolymer copolymer of of aa quaternized quaternized di- di-

C1-C4-alkyl-aminoethyl acrylate or methacrylate; POLYQUADTM; a polyaminoamide; and the the like. like.

[00298] Such formulations may be preferred for the introduction of pharmaceutically

acceptable formulations of any of the nucleic acids, engineered proteins, chemical agents,

antibodies, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia

virus, retrovirus, herpes virus, or AAV) disclosed herein. The formation and use of

liposomes is generally known to those of skill in the art. Recently, liposomes were developed

with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further,

various methods of liposome and liposome like preparations as potential drug carriers have

been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

[00299] Liposomes have been used successfully with a number of cell types that are

normally resistant to transfection by other procedures. In addition, liposomes are free of the

DNA length constraints that are typical of viral-based delivery systems. Liposomes have been

used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription

factors and allosteric effectors into a variety of cultured cell lines and animals. In addition,

several successful clinical trials examining the effectiveness of liposome-mediated drug

delivery have been completed.

[00300] Liposomes Liposomes are are formed formed fromfrom phospholipids phospholipids thatthat are are dispersed dispersed in aqueous in an an aqueous

medium and spontaneously form multilamellar concentric bilayer vesicles (also termed

multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 um. µm.

Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with

diameters in the range of 200 to 500 .ANG., containing an aqueous solution in the core.

Alternatively,

[00301] Alternatively, nanocapsule formulations nanocapsule formulations ofofthe recombinant the virus recombinant (e.g., virus lentivirus, (e.g., lentivirus,

alphavirus, vaccinia virus, adenovirus, retrovirus, herpes virus, or AAV) may be used.

Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side

effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1

um) should be designed using polymers able to be degraded in vivo. Biodegradable µm)

polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

WO wo 2020/069373 PCT/US2019/053545

Kits and Related Compositions

[00302] Any of the nucleic acids, engineered proteins, chemical agents, antibodies, and/or

recombinant viruses described herein may, in some embodiments, be assembled into

pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic

or research applications. A kit may include one or more containers housing the components

of the disclosure and instructions for use. Specifically, such kits may include one or more

agents described herein, along with instructions describing the intended application and the

proper use of these agents. In certain embodiments agents in a kit may be in a

pharmaceutical formulation and dosage suitable for a particular application and for a method

of administration of the agents. Kits for research purposes may contain the components in

appropriate concentrations or quantities for running various experiments.

In some

[00303] In some embodiments,the embodiments, the instant instant disclosure disclosurerelates to atokit relates for producing a kit a for producing a

virus, or AAV) and/or engineered cells, the kit comprising a container housing an engineered

nucleic acid (e.g., engineered nucleic acid) encoding OCT4, KLF4, SOX2, or a combination

thereof and/or host cells. In some embodiments, the kit further comprises instructions for

producing the recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus, adenovirus,

retrovirus, herpes virus, or AAV) and/or instructions for producing engineered cells. In some

embodiments, the kit further comprises at least one container housing a recombinant AAV

vector, wherein the recombinant AAV vector comprises a transgene (e.g., a gene associated

with ocular disease, such as corneal disease).

In some

[00304] In some embodiments,the embodiments, the instant instant disclosure disclosurerelates to atokit relates comprising a kit a comprising a

container housing any of the engineered nucleic acids (e.g., expression vectors), chemical

agents, antibodies, engineered cells, or recombinant viruses described herein. For example,

an expression vector or recombinant virus encoding KLF4, SOX2, OCT4, or a combination

thereof may comprise a sequence that is at least 70% (e.g., at least 75%, 80%, 85%, 90%,

95%, 98%, 99%, or 100%) identical to SEQ ID NO: 16, SEQ ID NO: 105, or SEQ ID NO:

121. In some embodiments, an expression vector or recombinant virus encoding KLF4,

SOX2, OCT4, or a combination thereof comprises SEQ ID NO: 16, SEQ ID NO: 105, or

SEQ ID NO: 121. In some embodiments, the expression vector encoding these three

transcription factors consists of SEQ ID NO: 16, SEQ ID NO: 105, or SEQ ID NO: 121. The

kit may further comprise an expression vector or recombinant virus encoding an inducing

agent. In some embodiments, an expression vector encoding an inducing agent comprises

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

SEQ ID NO: 17, SEQ ID NO: 31, or SEQ ID NO: 32. In some embodiments, the expression

vector encoding an inducing agent consists of SEQ ID NO: 17, SEQ ID NO: 31, or SEQ ID

NO: 32. See, e.g., U.S. Provisional Application No. 62/738,894, entitled MUTANT

REVERSE TETRACYCLINE TRANSACTIVATORS FOR EXPRESSION OF GENES, which was filed on September 28, 2018, under attorney docket number

H0824.70300US00,and isherein H0824.70300US00,: and is herein incorporated incorporated by reference by reference in itsin its entirety. entirety.

[00305] The The kit kit may may be designed be designed to facilitate to facilitate use use of the of the methods methods described described herein herein by by

researchers and can take many forms. Each of the compositions of the kit, where applicable,

may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In

certain cases, some of the compositions may be constitutable or otherwise processable (e.g.,

to an active form), for example, by the addition of a suitable solvent or other species (for

example, water or a cell culture medium), which may or may not be provided with the kit.

As used herein, "instructions" can define a component of instruction and/or promotion, and

typically involve written instructions on or associated with packaging of the disclosure.

Instructions also can include any oral or electronic instructions provided in any manner such

that a user will clearly recognize that the instructions are to be associated with the kit, for

example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based

communications, etc. The written instructions may be in a form prescribed by a

governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological

products, which instructions can also reflect approval by the agency of manufacture, use or

sale for animal administration.

[00306] The The kit kit may may contain contain any any one one or more or more of the of the components components described described herein herein in one in one

or more containers. As an example, in one embodiment, the kit may include instructions for

mixing one or more components of the kit and/or isolating and mixing a sample and applying

to a subject. The kit may include a container housing agents described herein. The agents

may be in the form of a liquid, gel or solid (powder). The agents may be prepared sterilely,

packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or or

other container for storage. A second container may have other agents prepared sterilely.

Alternatively the kit may include the active agents premixed and shipped in a syringe, vial,

tube, or other container. The kit may have one or more or all of the components required to

administer the agents to an animal, such as a syringe, topical application devices, or iv needle

tubing and bag, particularly in the case of the kits for producing specific somatic animal

models.

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

[00307] The The kit kit may may havehave a variety a variety of forms, of forms, suchsuch as aasblister a blister pouch, pouch, a shrink-wrapped a shrink-wrapped

pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray

form, with the accessories loosely packed within the pouch, one or more tubes, containers, a

box or a bag. The kit may be sterilized after the accessories are added, thereby allowing the

individual accessories in the container to be otherwise unwrapped. The kits can be sterilized

using any appropriate sterilization techniques, such as radiation sterilization, heat

sterilization, or other sterilization methods known in the art. The kit may also include other

components, depending on the specific application, for example, containers, cell media, salts,

buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a

disinfecting agent, disposable gloves, a support for the agents prior to administration etc.

[00308] The The instructions instructions included included within within the the kit kit may may involve involve methods methods for for detecting detecting a a

latent AAV in a cell. In addition, kits of the disclosure may include, instructions, a negative

and/or positive control, containers, diluents and buffers for the sample, sample preparation

tubes and a printed or electronic table of reference AAV sequence for sequence comparisons.

Therapeutic Applications

[00309] Any Any of the of the nucleicacids nucleic acids (e.g., (e.g., engineered engineerednucleic acids) nucleic capable acids) of inducing capable of inducing

OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), engineered cells, engineered

retrovirus, herpes virus, or AAV) described herein may be used for regulating (e.g., inducing

or inducing and stopping) cellular reprogramming, tissue repair, tissue regeneration, organ

regeneration, reversing aging, treating a disease, or any combination thereof. Any of the

nucleic acids (e.g., engineered nucleic acids) capable of inducing expression of a combination

of transcription factors selected from OCT4, KLF4, and SOX2 (e.g., OCT4 and KLF4, OCT4

and SOX2, SOX2 and KLF4, or KLF4, OCT4, and SOX2), engineered cells, engineered

proteins, chemical agents activating (e.g., inducing expression of) a combination of

transcription factors selected from OCT4, KLF4, and SOX2 (e.g., OCT4 and KLF4, OCT4

and SOX2, SOX2 and KLF4, or KLF4, OCT4, and SOX2), antibodies activating (e.g.,

inducing expression of) combination of transcription factors selected from OCT4, KLF4, and

SOX2 (e.g., OCT4 and KLF4, OCT4 and SOX2, SOX2 and KLF4, or KLF4, OCT4, and

SOX2), and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus,

WO wo 2020/069373 PCT/US2019/053545

regeneration, regeneration,reversing aging, reversing treating aging, a disease, treating or any combination a disease, thereof. In or any combination some thereof. In some

embodiments, any of the nucleic acid (e.g., engineered nucleic acid) capable of inducing

OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), any of the engineered cells,

any of the engineered proteins, any of the chemical agents activating (e.g., inducing

expression of) OCT4, KLF4, and/or SOX2, any of the antibodies activating (e.g., inducing

expression of) OCT4, KLF4, and/or SOX2, and/or any of the recombinant viruses (e.g.,

lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) may be

useful in regulating cellular reprogramming, tissue repair, tissue survival, tissue regeneration,

tissue growth, tissue function, organ regeneration, organ survival, organ function, or any

combination thereof, optionally wherein regulating comprises inducing cellular

repair, tissue survival, tissue regeneration, tissue growth, angiogenesis, scar formation, the

appearance of aging, organ regeneration, organ survival, altering the taste and quality of

agricultural products derived from animals, treating a disease, or any combination thereof, in

vivo or in vitro may be administered to a cell, tissue, or organ that is in vivo (e.g., part of a

subject), or may be administered to a cell, tissue, or organ ex vivo. In some embodiments,

any of the nucleic acid (e.g., engineered nucleic acid) (e.g., expression vector) capable of

inducing expression of OCT4; KLF4; SOX2; or any combination thereof thereof,any anyof ofthe the

engineered cells, any of the engineered proteins, any of the chemical agents activating (e.g.,

inducing expression of) OCT4; KLF4; SOX2; or any combination thereof, any of the

antibodies activating (e.g., inducing expression of) OCT4, KLF4, SOX2, or a combination

vaccinia virus, retrovirus, herpes virus, or AAV) may be useful in regulating cellular

reprogramming, tissue repair, tissue survival, tissue regeneration, tissue growth, tissue

function, organ regeneration, organ survival, organ function, or any combination thereof,

optionally wherein regulating comprises inducing cellular reprogramming, reversing aging,

improving tissue function, improving organ function, tissue repair, tissue survival, tissue

regeneration, tissue growth, angiogenesis, scar formation, the appearance of aging, organ

regeneration, organ survival, altering the taste and quality of agricultural products derived

from animals, treating a disease, or any combination thereof, in vivo or in vitro may be

administered to a cell, tissue, or organ that is in vivo (e.g., part of a subject), or may be

administered to a cell, tissue, or organ ex vivo. As used herein, regulating may refer to any

WO wo 2020/069373 PCT/US2019/053545

type of modulation, including inducing or promoting, inhibiting, and/or stopping.

Angiogenesis refers to growth of new blood vessels, including capillaries.

In instances,

[00310] In some

[00310] some instances, a viral a viral vectorvector (e.g.,(e.g., lentivirus lentivirus vector, vector, alphavirus alphavirus vector, vector,

vaccinia virus vector, adenovirus vector, herpes virus vector, retrovirus vector, or AAV

vector) is administered in a recombinant virus (e.g., lentivirus, alphavirus, vaccinia virus,

adenovirus, herpes virus, retrovirus, or AAV). Without being bound by a particular theory,

transient expression of OCT4, SOX2, and KLF4 may result in partial reprogramming of a

cell. For example, partial reprogramming may induce a fully differentiated cell to rejuvenate

and gain pluripotency. In some embodiments, transient expression of OCT4, SOX2, and/or

KLF4 does not induce expression of stem cell markers (e.g., Nanog).

In some

[00311] In some embodiments, embodiments, transient transient expression expression of OCT4, of OCT4, SOX2, SOX2, KLF4, KLF4, or aor a

combination thereof does not induce expression of stem cell markers (e.g., Nanog). Without

being bound by any particular theory, Nanog activation may induce teratomas and cause

death of the host. In some embodiments, the method does not induce teratoma formation. In

some embodiments, the method does not induce unwanted cell proliferation. In some

embodiments, the method does not induce malignant cell growth. In some embodiments, the

method does not induce cancer. In some embodiments, the method does not induce

glaucoma. In some embodiments, transient expression is at most 1 hour, 5 hours, 24 hours, 2

days, 3 days, 4 days, 5, days, or 1 week. In some instances, prolonged expression (e.g.,

continued expression for at least 5 days, at least 1 week, or at least 1 month) of OCT4, SOX2,

and KLF4, results in full reprogramming of a cell. For example, a cell may be fully

reprogrammed into a pluripotent cell (e.g., induced pluripotent cell). In some instances,

prolonged expression (e.g., continued expression for at least 5 days, at least 1 week, or at

least 1 month) of OCT4, SOX2, KLF4, or a combination thereof, results in full

reprogramming of a cell. For example, a cell may be fully reprogrammed into a pluripotent

cell (e.g., induced pluripotent cell).

Without

[00312] Without being being bound bound by abyparticular a particular theory, theory, expression expression of OCT4, of OCT4, SOX2, SOX2, and and

KLF4 may promote cellular reprogramming, promote tissue regeneration, promote organ

regeneration, reverse aging, treat a disease, or any combination thereof because OCT4,

SOX2, and KLF4 induce partial reprogramming. As used herein, partial or incomplete

reprogramming of a cell refers to a cell that are not stem cells, but have youthful

characteristics. In some embodiments, a youthful characteristic is an epigenome that is

similar to a young cell. In some embodiments, a stem cell shows higher levels of Nanog

expression compared to a cell that is not a stem cell. In some embodiments, youthful

WO wo 2020/069373 PCT/US2019/053545

characteristics refers to rejuvenation of a cell without changing cell identity. See, e.g.,

shown in FIG.16, in which the expression of histone and Chaf (Chromatin assembly factor)

genes decline during aging in ear fibroblasts from aged mice (12 months or 15 months)

compared to those from young mice, short term of OSKM (3 days) or OSK expression (5

days) induction can reset their gene expression level to young state, without making the cells

into a stem cell (e.g., Nanog expression is not induced in these cells).

[00313] To practice To practice thisthis embodiment, embodiment, an effective an effective amount amount of any of any of the of the nucleic nucleic acids acids

(e.g., engineered nucleic acids) capable of inducing OCT4, KLF4, and/or SOX2 expression

(e.g., expression vector), engineered proteins, chemical agents activating (e.g., inducing

expression of) OCT4, KLF4, and/or SOX2, antibodies activating (e.g., inducing expression

of) OCT4, KLF4, and/or SOX2, and/or recombinant viruses (e.g., lentivirus, adenovirus,

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) are administered to a cell, a

tissue, organ, and/or subject. In some embodiments, an effective amount of any of the

nucleic acids (e.g., engineered nucleic acids) (e.g., expression vector) capable of inducing

expression of OCT4, KLF4, SOX2, or a combination thereof, engineered proteins, chemical

agents activating (e.g., inducing expression of) OCT4, KLF4, SOX2, or a combination

thereof, antibodies activating (e.g., inducing expression of) OCT4, KLF4, SOX2, or a

vaccinia virus, retrovirus, herpes virus, or AAV) are administered to a cell, a tissue, organ,

and/or subject. Engineered cells may be administered to any tissue, organ, and/or subject.

When the expression vector comprises an inducible promoter (e.g., a TRE promoter,

including a TRE3G, TRE2, or P tight), the inducing agent may also be introduced into the

cell (e.g., simultaneously or sequentially with one or more nucleic acids (e.g., engineered

nucleic acids) encoding OCT4, SOX2, KLF4, or any combination thereof). In one

embodiment, OCT4, SOX2, and KLF4 are encoded by one expression vector that is separate

from an expression vector encoding the inducing agent. In some instances, the inducing

agent is encoded by the same expression vector that encodes OCT4, SOX2, KLF4, or any

combination thereof.

In some

[00314] In some instances,ananinducing instances, inducing agent agent(e.g., (e.g.,a nucleic acidacid a nucleic encoding an inducing encoding an inducing

agent, an engineered protein encoding an inducing agent, or a virus encoding an inducing

agent) and/or a chemical agent (e.g., tetracycline) that is capable of modulating (e.g.,

activating or inhibiting) activity of the inducing agent is also introduced into a cell, tissue,

organ, and/or subject. In certain embodiments, a cell, tissue, subject, and/or organ is further

cultured in the presence or absence of a chemical agent that is capable of modulating the

120

WO wo 2020/069373 PCT/US2019/053545

activity of an inducing agent (e.g., tetracycline, which includes doxycycline). For a Tet-On

system, the inducing agent may be rtTA (e.g., rtTA3 or rtTA4), and the inducing agent

promotes expression of OCT4, SOX2, KLF4, or any combination thereof in the presence of

tetracycline. For a Tet-Off system, the inducing agent may be tTA, and the inducing agent

promotes expression of OCT4, SOX2, KLF4, or any combination thereof in the absence of

tetracycline.

[00315] Administration Administration of expression of an an expression vector vector encoding encoding a transcription a transcription factor factor described described

herein and in some cases the inducing agent (e.g., a nucleic acid (e.g., engineered nucleic

acid) encoding an inducing agent or the inducing agent as protein) and/or chemical agent that

is capable of modulating the activity of the inducing agent under suitable conditions for

expression may increase expression of the transgene by at least 10%, at least 20%, at least

30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least

100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 500%, at least

600%, at least 700%, at least 800%, at least 900%, or at least 1,000% in a cell. Gene

expression may be determined by routine methods including enzyme-linked immunosorbent

assays (ELISAs), western blots, and quantification of RNA (e.g., reverse transcription

polymerase chain reaction).

[00316] In some In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acid) acid)

capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector),

engineered proteins described herein, chemical agents activating (e.g., inducing expression

of) OCT4, KLF4, and/or SOX2, antibodies activating (e.g., inducing expression of) OCT4,

KLF4, and/or SOX2, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

may be introduced to a tissue, cell, or organ ex vivo (e.g., not in a subject) and the tissue, cell,

and/or organ may be further cultured ex vivo. In some embodiments, any of the nucleic acids

(e.g., engineered nucleic acid) (e.g., expression vector) capable of inducing expression of

OCT4; KLF4; SOX2; or any combination thereof, engineered proteins described herein,

combination thereof, antibodies activating (e.g., inducing expression of) OCT4; KLF4;

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) described herein, alone, or in

combination may be introduced to a tissue, cell, or organ ex vivo (e.g., not in a subject) and

the tissue, cell, and/or organ may be further cultured ex vivo. In some instances, an inducing

agent and/or a chemical agent capable of modulating the activity of the inducing agent is

WO wo 2020/069373 PCT/US2019/053545 PCT/US2019/053545

introduced to a tissue, cell, and/or organ ex vivo and the tissue, cell, and/or organ may be

further cultured ex vivo. In some embodiments, engineered cells are cultured to produce an

engineered tissue. In some embodiments, engineered cells are cultured to produce an

engineered organ. In some embodiments, an engineered tissue is cultured to produce an

engineered organ. These methods may be useful in producing an engineered (e.g., eye, ear,

brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine) cell,

engineered (e.g., eye, ear, nose, mouth including gum and roots of teeth, bone, lung, breast,

or intestine) tissue or organ (e.g., eye, ear, nose, mouth including gum and roots of teeth,

penis, cloaca, fin, ovary, or intestine) for transplantation into a subject. In some

embodiments, the engineered cell, tissue, and/or organ is transplanted into a subject.

[00317] In some embodiments, cells, tissues, organs, or any combination thereof to be

engineered are autologous to the subject, e.g., obtained from a subject in need thereof.

Administration of autologous cells, autologous tissues, autologous organs, or any

combination thereof may result in reduced rejection of the cells, tissues, organs, or any

combination thereof compared to administration of non-autologous cells, non-autologous

tissue and/or non-autologous organs. Alternatively, the cells, tissues, or organs to be

engineered maybebe engineered may allogenic allogenic cells, cells, allogenic allogenic tissues, tissues, or allogenic or allogenic organs. organs. For For example, example,

allogenic cells, allogenic tissue, allogenic organs, or any combination thereof may be derived

from a donor (e.g., from a particular species) and administered to a recipient (e.g., from the

same species) who is different from the donor. In some embodiments, allogenic cells,

allogenic tissue, allogenic organs, or any combination thereof may be derived from a donor

subject from a particular species and administered to a recipient subject from a different

species from the donor.

[00318] In some embodiments, the engineered cell is a stem cell (iPSC) including naive naïve

iPSC that can different into three germ layers. In some embodiments, the iPSC is further

differentiated into another cell type (e.g., eye, ear, nose, mouth including gum and roots of

teeth, bone, lung, breast, udder, pancreas, stomach, oesophagus, muscle including cardiac

WO wo 2020/069373 PCT/US2019/053545

prostate, penis, cloaca, fin, ovary, or intestine). The iPSC may be further differentiated using

methods known in the art (e.g., ex vivo)

[00319] In some In some embodiments, embodiments, engineered engineered cells cells comprise comprise moremore thanthan one one cellcell typetype (e.g., (e.g.,

[00320] As aAsnon-limiting a non-limiting example, example, any any of the of the engineered engineered nucleic nucleic acids acids (e.g., (e.g., naked naked

nucleic acids or nucleic acids formulated in a delivery vehicle, including a viral vector and/or

nanoparticle) encoding OCT4, KLF4, and SOX2, may be delivered to a cell (e.g., a

differentiated cell) to produce an induced pluripotent stem cell. In some embodiments, the

induced pluripotent stem cell is further differentiated (e.g., differentiated into an eye, ear,

brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine cell). In

some embodiments, cells are engineered ex vivo and administered to a subject in need

thereof. In some embodiments, an organ or a tissue may be regenerated in vitro using iPSCs

and the organ or tissue is transplanted into an individual.

[00321] As aAsnon-limiting a non-limiting example, example, the the methods methods described described herein herein may may be used be used to produce to produce

engineered skin, an engineered liver, an engineered eye, an engineered liver, any engineered

cell, any engineered organ, or any engineered tissue ex vivo. The engineered organ,

engineered tissue, engineered organ, or any combination thereof may be administered to a

subject. In some embodiments, administration of an engineered cell, engineered tissue,

engineered organ, or a combination thereof improves survival of a subject (e.g., increases the

lifespan of a subject relative to not receiving the engineered cell, tissue, or organ).

[00322] A pharmaceutical composition described herein may be administered to a subject

in need thereof. Non-limiting examples of subjects include any animal (e.g., mammals,

including humans). A subject may be suspected of having, be at risk for or have a condition.

For example, the condition may be an injury or a disease and the condition may affect any

tissue (e.g., ear, nose, mouth including gum and roots of teeth, bone, lung, breast, udder,

intestine). Non-limiting examples of conditions, diseases, and disorders include acute

injuries, neurodegenerative disease, chronic diseases, proliferative diseases, cardiovascular

diseases, genetic diseases, inflammatory diseases, autoimmunue diseases, neurological

WO wo 2020/069373 PCT/US2019/053545

diseases, hematological diseases, painful conditions, psychiatric disorders, metabolic

disorders, cancers, aging, age-related diseases, and diseases affecting any tissue in a subject.

In some embodiments, the disease is an ocular disease.

In certain

[00323] In certain embodiments, any embodiments, any of of the the nucleic nucleicacids (e.g., acids engineered (e.g., nucleic engineered acid) acid) nucleic

may be introduced to a subject prior to the onset of a disease (e.g., to prevent a disease or to

prevent damage to a cell, tissue, or organ). In certain embodiments, any of the nucleic acids

combination may be introduced to a subject prior to the onset of a disease (e.g., to prevent a

disease or to prevent damage to a cell, tissue, or organ). In certain embodiments, an inducing

agent and/or a chemical agent capable of modulating activity of the inducing agent may be

introduced to a subject prior to the onset of a disease. In some embodiments, the subject may

be a healthy subject. In certain embodiments, any of the nucleic acids (e.g., engineered

nucleic acid) capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression

vector), engineered proteins described herein, chemical agents activating (e.g., inducing

combination may be introduced to a subject following the onset of disease (e.g., to alleviate

the damage or symptoms associated with a disease). In certain embodiments, any of the

nucleic acids (e.g., engineered nucleic acid) capable of inducing expression of OCT4; KLF4;

SOX2; or any combination thereof expression, engineered proteins described herein,

PCT/US2019/053545

combination, may be introduced to a subject following the onset of disease (e.g., to alleviate

the damage or symptoms associated with a disease). In some embodiments, OCT4, KLF4,

and/or SOX2 expression is induced prior to the onset of a disease. In some embodiments,

expression of OCT4; KLF4; SOX2; or any combination thereof is induced prior to the onset

of a disease. In some embodiments, OCT4, KLF4, and/or SOX2 expression is induced after

the onset of a disease. In some embodiments, expression of OCT4; KLF4; SOX2; or any

combination thereof is induced after the onset of a disease. In some embodiments, OCT4,

KLF4, and/or SOX2 expression is induced in a young subject, young cell, young tissue,

and/or young organ. In some embodiments, OCT4, KLF4, and/or SOX2 expression is

induced in an aged subject, aged cell, aged tissue, and/or aged organ. In some embodiments,

expression of OCT4; KLF4; SOX2; or any combination thereof is induced in a young subject,

young cell, young tissue, and/or young organ. In some embodiments expression of of,OCT4; OCT4;

KLF4; SOX2; or any combination thereof is induced in an aged subject, aged cell, aged

tissue, and/or aged organ. In certain embodiments, an inducing agent and/or a chemical agent

capable of modulating activity of the inducing agent may be introduced to a subject following

the onset of a disease.

[00324] In certain In certain embodiments, embodiments, the the tissue tissue may may be considered be considered healthy healthy but but suboptimal suboptimal for for

performance or survival in current or future conditions (e.g., in agriculture, or adverse

conditions including disease treatments, toxic therapies, sun exposure, or space travel outside

the the earth's earth'satmosphere). atmosphere).

In certain

[00325] In certain embodiments, embodiments, the the condition condition is aging. is aging. All All animals animals typically typically go through go through

a period of growth and maturation followed by a period of progressive and irreversible

physiological decline ending in death. The length of time from birth to death is known as the

life span of an organism, and each organism has a characteristic average life span. Aging is a

physical manifestation of the changes underlying the passage of time as measured by percent

of average life span.

In some

[00326] In some cases, cases, characteristics of characteristics of aging agingcan canbebe quite obvious. quite For example, obvious. For example,

characteristics of older humans include skin wrinkling, graying of the hair, baldness, and

cataracts, as well as hypermelanosis, osteoporosis, cerebral cortical atrophy, lymphoid

depletion, thymic atrophy, increased incidence of diabetes type II, atherosclerosis, cancer,

and heart disease. Nehlin et al. (2000), Annals NY Acad Sci 980:176-79. Other aspects of

mammalian aging include weight loss, lordokyphosis (hunchback spine), absence of vigor,

lymphoid atrophy, decreased bone density, dermal thickening and subcutaneous adipose

WO wo 2020/069373 PCT/US2019/053545

tissue, decreased ability to tolerate stress (including heat or cold, wounding, anesthesia, and

hematopoietic precursor cell ablation), liver pathology, atrophy of intestinal villi, skin

ulceration, amyloid deposits, and joint diseases. Tyner et al. (2002), Nature 415:45-53.

Those

[00327] Those skilled skilled ininthe theart art will will recognize recognizethat thethe that aging process aging is also process is manifested at also manifested at

the cellular level, as well as in mitochondria. Cellular aging is manifested in loss of doubling

capacity, increased levels of apoptosis, changes in differentiated phenotype, and changes in

metabolism, e.g., decreased levels of protein synthesis and turnover.

[00328] Given the programmed nature of cellular and organismal aging, it is possible to

evaluate the "biological age" of a cell or organism by means of phenotypic characteristics

that are correlated with aging. For example, biological age can be deduced from patterns of

gene expression, resistance to stress (e.g., oxidative or genotoxic stress), rate of cellular

proliferation, and the metabolic characteristics of cells (e.g., rates of protein synthesis and

turnover, mitochondrial function, ubiquinone biosynthesis, cholesterol biosynthesis, ATP

levels within the cell, levels of a Krebs cycle intermediate in the cell, glucose metabolism,

nucleic acid (e.g., engineered nucleic acid) metabolism, ribosomal translation rates, etc.). As

used herein, "biological age" is a measure of the age of a cell or organism based upon the

molecular characteristics of the cell or organism. Biological age is distinct from "temporal

age," which age," whichrefers to to refers the the age age of a of cell or organism a cell as measured or organism by days, months, as measured by days,andmonths, years. and years.

[00329] The The raterate of of agingofofan aging an organism, organism, e.g., e.g.,ananinvertebrate (e.g., invertebrate a worm (e.g., a or a fly) worm or aorfly) a or a

vertebrate (e.g., a rodent, e.g., a mouse) can be determined by a variety of methods, e.g., by

one or more of: a) assessing the life span of the cell or the organism; (b) assessing the

presence or abundance of a gene transcript or gene product in the cell or organism that has a

biological age-dependent expression pattern; (c) evaluating resistance of the cell or organism

to stress, e.g., genotoxic stress (e.g., etoposide, UV irradiation, exposure to a mutagen, and SO so

forth) or oxidative stress; (d) evaluating one or more metabolic parameters of the cell or

organism; (e) evaluating the proliferative capacity of the cell or a set of cells present in the

organism; and (f) evaluating physical appearance or behavior of the cell or organism. In one

example, evaluating the rate of aging includes directly measuring the average life span of a

group of animals (e.g., a group of genetically matched animals) and comparing the resulting

average to the average life span of a control group of animals (e.g., a group of animals that

did did not notreceive receivethethe test compound test but are compound butgenetically matched matched are genetically to the group of animals to the that group of did animals that did

receive the test compound). Alternatively, the rate of aging of an organism can be determined

by measuring an age-related parameter. Examples of age-related parameters include:

appearance, e.g., visible signs of age; the expression of one or more genes or proteins (e.g.,

WO wo 2020/069373 PCT/US2019/053545

genes or proteins that have an age-related expression pattern); resistance to oxidative stress;

metabolic parameters (e.g., protein synthesis or degradation, ubiquinone biosynthesis,

cholesterol biosynthesis, ATP levels, glucose metabolism, nucleic acid (e.g., engineered

nucleic acid) metabolism, ribosomal translation rates, etc.); and cellular proliferation (e.g., of

retinal cells, bone cells, white blood cells, etc.).

[00330] Aging Aging can can alsoalso be determined be determined by the by the raterate of change of change of biomarkers of biomarkers (e.g., (e.g.,

epigenetic marks including DNA methylation level of CpG island in the genome (known as

the "Horvath Clock") beta-galactosidase-positive cells in cells, gene expression changes, or

certain changes to the abundance of molecules in the bloodstream). An example is an

algorithm from Segterra Inc. that determines "InnerAge" based on blood biomarkers (see

InsideTracker.com).

As shown

[00331] As shown in the in the Examples Examples herein, herein, recombinant recombinant viruses viruses (e.g., (e.g., AAVs) AAVs) encoding encoding

OCT4, KLF4, and SOX2 promoted regeneration of axons, which may be used to prevent or

alleviate neurodegeneration that is often associated with aging. The methods may be used to

prevent or alleviate neurodegeneration and peripheral neuropathies associated.

Neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, multiple

sclerosis, amniotropic lateral sclerosis (ALS), Huntington's disease, and muscular dystrophy.

Neurodegeneration may be quantified using any method known in the art. For example, the

executive function of an individual may be determined (Moreira et al., Front Aging Neurosci.

2017 Nov 9;9:369).

In some

[00332] In some embodiments, embodiments, expression, expression, induction, induction, or activation or activation of OCT4, of OCT4, SOX2, SOX2,

KLF4, or a combination thereof as described herein increases the number of axons per nerve

in a tissue, organ, or a subject relative to a control. In some embodiments, a method

described herein increases the number of axons per nerve by at least 1.5 fold, by at least 2

fold, by at least 3 fold, by at least 5 fold, by at least 6 fold, by at least 7 fold, by at least 8

fold, by at least 9 fold, by at least 10 fold, by at least 20 fold, by at least 30 fold, by at least 40

fold, by at least 50 fold, by at least 60 fold, by at least 70 fold, by at least 80 fold, by at least

90 fold, or by at least 100 fold relative to a control. In some embodiments, the control is the

number of axons per nerve in the tissue, organ, or subject prior to expression, induction, or

activation of OCT4, SOX2, KLF4, or a combination thereof.

Additional

[00333] Additional age-relatedconditions age-related conditions which whichmay maybebe treated include treated heartheart include failure, failure,

stroke, diabetes, liver diseases, fibrotic diseases, osteoporosis, arthritis, hearing loss (partial

or total), eye-related conditions (e.g., poor eye sight, retinal disease, any ocular disease (e.g.,

any condition affecting the eye)), glaucoma, muscle diseases (e.g., sarcopenia and muscular

WO wo 2020/069373 PCT/US2019/053545

dystrophies), frailty, a progeroid syndrome (e.g., Hutchinson-Gilford progeria syndrome),

and cancer. In certain embodiments, the disease is a retinal disease (e.g., macular

degeneration).

[00334] In some In some embodiments, embodiments, expression, expression, induction, induction, or activation or activation of OCT4, of OCT4, SOX2, SOX2,

KLF4, or a combination thereof in a neuron increases neurite area of the neuron by at least

1.5 fold, by at least 2 fold, by at least 3 fold, by at least 5 fold, by at least 6 fold, by at least 7

fold, by at least 8 fold, by at least 9 fold, by at least 10 fold, by at least 20 fold, by at least 30

fold, by at least 40 fold, by at least 50 fold, by at least 60 fold, by at least 70 fold, by at least

80 fold, by at least 90 fold, or by at least 100 fold relative to the neuron without expression,

induction, or activation of OCT4, SOX2, KLF4, or a combination thereof.

In some

[00335] In some embodiments, embodiments, expression, expression, induction, induction, or activation or activation of OCT4, of OCT4, SOX2, SOX2,

KLF4, or a combination thereof as described herein increases the axon density in a tissue,

organ, or a subject relative to a control. In some embodiments, a method described herein

increases axon density at least 1.5 fold, by at least 2 fold, by at least 3 fold, by at least 5 fold,

by at least 6 fold, by at least 7 fold, by at least 8 fold, by at least 9 fold, by at least 10 fold, by

at least 20 fold, by at least 30 fold, by at least 40 fold, by at least 50 fold, by at least 60 fold,

by at least 70 fold, by at least 80 fold, by at least 90 fold, or by at least 100 fold relative to a

control. In some embodiments, the control is the axon density in the tissue, organ, or subject

prior to expression, induction, or activation of OCT4, SOX2, KLF4, or a combination

thereof.

[00336] In some In some embodiments, embodiments, expression, expression, induction, induction, or activation or activation of OCT4, of OCT4, SOX2, SOX2,

KLF4, or a combination thereof in a subject increases the visual acuity of the subject relative

to a control. In some embodiments, a method described herein increases the visual acuity of

a subject by at least 1.5 fold, by at least 2 fold, by at least 3 fold, by at least 5 fold, by at least

6 fold, by at least 7 fold, by at least 8 fold, by at least 9 fold, by at least 10 fold, by at least 20

fold, by at least 30 fold, by at least 40 fold, by at least 50 fold, by at least 60 fold, by at least

70 fold, by at least 80 fold, by at least 90 fold, or by at least 100 fold relative to a control. In

some embodiments, the control is the visual acuity of the subject prior to expression,

induction, or activation of OCT4, SOX2, KLF4, or a combination thereof. In some

embodiments, visual acuity is measured by optomotor acuity. In some embodiments, visual

acuity is measured using a pattern electroretinogram response. In some emboidments, visual

acuity is measured using a distance visual acuity test, which may include the use of a Snellen

chart or E chart. See, e.g., Marsden et al., Community Eye Health. 2014; 27(85): 16 and the

Examples below.

WO wo 2020/069373 PCT/US2019/053545

[00337] In some embodiments, expression, induction, or activation of OCT4, SOX2,

KLF4, or a combination thereof in a subject decreases the intraocular pressure of the subject

relative to a control. In some embodiments, a method described herein decreases the

intraocular pressure of a subject by at least 1.5 fold, by at least 2 fold, by at least 3 fold, by at

least 5 fold, by at least 6 fold, by at least 7 fold, by at least 8 fold, by at least 9 fold, by at

least 10 fold, by at least 20 fold, by at least 30 fold, by at least 40 fold, by at least 50 fold, by

at least 60 fold, by at least 70 fold, by at least 80 fold, by at least 90 fold, or by at least 100

fold relative to a control. In some embodiments, the control is the intraocular pressure of the

subject prior to expression, induction, or activation of OCT4, SOX2, KLF4, or a combination

thereof. See, e.g., the Examples below.

In some

[00338] In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

vaccinia virus, retrovirus, herpes virus, or AAV) described herein may be used to treat and/or

prevent any of the diseases described herein. In some embodiments, an inducing agent and/or

a chemical agent capable of modulating activity of the inducing agent is also used.

[00339] As aAsnon-limiting a non-limiting example, example, an engineered an engineered cellcell of the of the present present disclosure disclosure may may be be

used to replace a dysfunctional cell in a subject in need thereof. As another non-limiting

example, any of the nucleic acids (e.g., engineered nucleic acids) capable of inducing OCT4,

KLF4, and/or SOX2 expression (e.g., expression vector), nucleic acids (e.g., engineered

nucleic acids) (e.g., expression vector) capable of inducing expression of a combination of at

least two (e.g., at least three) transcription factors selected from OCT4, KLF4, and SOX2,

and/or SOX2, chemical agents activating (e.g., inducing expression of) a combination of at

least least two two(e.g., (e.g.,at at least three) least transcription three) factorsfactors transcription selectedselected from OCT4,from KLF4, and SOX2, OCT4, KLF4, and SOX2,

antibodies activating (e.g., inducing expression of) OCT4, KLF4, and/or SOX2, antibodies

activating (e.g., inducing expression of) a combination of at least two (e.g., at least three)

transcription factors selected from OCT4, KLF4, and SOX2, and/or recombinant viruses

may be used to (e.g., incompletely or fully) reprogram a cell in vivo or in vitro. In some

embodiments, an inducing agent and/or a chemical agent capable of modulating activity of

the inducing agent is also used. For example, any of the any of the nucleic acids (e.g.,

PCT/US2019/053545

expression vector), engineered proteins, chemical agents activating (e.g., inducing expression

vaccinia virus, retrovirus, herpes virus, or AAV) may be used to produce an engineered cell

(e.g., an induced pluripotent stem cell). For example, any of the nucleic acids (e.g.,

engineered nucleic acids) (e.g., expression vector) capable of inducing expression of OCT4,

KLF4, SOX2, or a combination thereof, engineered proteins, chemical agents activating (e.g.,

inducing expression of) OCT4, KLF4, SOX2, or a combination thereof, antibodies activating

(e.g., inducing expression of) OCT4, KLF4, SOX2, or a combination thereof, and/or

herpes virus, or AAV) may be used to produce an engineered cell (e.g., an induced

pluripotent stem cell). The engineered cell (e.g., induced pluripotent stem cell) may then be

administered to a subject in need thereof. In some embodiments, the engineered cell is

cultured in the presence of an inducing agent and/or a chemical agent capable of modulating

activity of the inducing agent. In some embodiments, an inducing agent and/or a chemical

agent capable of modulating activity of the inducing agent is also administered to the subject.

Non-limiting

[00340] Non-limiting usesofofthe uses the nucleic nucleic acids acids(e.g., (e.g.,engineered nucleic engineered acids)acids) nucleic capablecapable of of

inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), nucleic acids (e.g.,

engineered nucleic acids) (e.g., expression vector) capable of inducing expression of a

combination of at least two (e.g., at least three) transcription factors selected from OCT4,

KLF4, and SOX2, engineered proteins, chemical agents activating (e.g., inducing expression

of) OCT4, KLF4, and/or SOX2, chemical agents activating (e.g., inducing expression of) a

KLF4, and SOX2, engineered cells, antibodies activating (e.g., inducing expression of)

OCT4, KLF4, and/or SOX2, antibodies activating (e.g., inducing expression of) a

KLF4, and SOX2, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus,

vaccinia virus, retrovirus, herpes virus, or AAV) include wound healing, bleed out, injuries,

broken bones, gunshot wounds, cuts, scarring during surgery (e.g., cesarean). In some

the inducing agent is also used.

[00341] In some In some embodiments, embodiments, any any of the of the of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic

acids) capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector),

WO wo 2020/069373 PCT/US2019/053545

expression of a combination of at least two (e.g., at least three) transcription factors selected

from OCT4, KLF4, and SOX2, engineered cells, engineered proteins, chemical agents

activating (e.g., inducing expression of) OCT4, an KLF4, and/or SOX2, chemical agents

transcription factors selected from OCT4, KLF4, and SOX2, antibodies activating (e.g.,

inducing expression of) OCT4, KLF4, and/or SOX2, antibodies activating (e.g., inducing

expression of) a combination of at least two (e.g., at least three) transcription factors selected

from OCT4, KLF4, and SOX2, and/or recombinant viruses (e.g., lentivirus, adenovirus,

alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) are used to treat disease that

affects a non-human subject (e.g., a disease affecting livestock, domesticated pets, and/or

other non-human animals). In some embodiments, an inducing agent and/or a chemical agent

capable of modulating activity of the inducing agent is also used. For example, the disease

may be a cattle disease, a primate (e.g., cynomolgus monkeys, rhesus monkeys) disease, a

disease affecting a commercially relevant animal, such as cattle, pigs, horses, sheep, goats,

cats, and/or dogs) and/or a disease affecting birds (e.g., commercially relevant birds, such as

chickens, ducks, geese, and/or turkeys).

In some

[00342] In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vector), nucleic

acids (e.g., engineered nucleic acids) (e.g., expression vector) capable of inducing expression

of a combination of at least two (e.g., at least three) transcription factors selected from OCT4,

KLF4, and SOX2, engineered cells, engineered proteins, chemical agents activating (e.g.,

inducing expression of) OCT4, KLF4, and/or SOX2, chemical agents activating (e.g.,

inducing expression of) a combination of at least two (e.g., at least three) transcription factors

selected from OCT4, KLF4, and SOX2, antibodies activating (e.g., inducing expression of)

OCT4, KLF4, and/or SOX2, antibodies activating (e.g., inducing expression of) a

vaccinia virus, retrovirus, herpes virus, or AAV) described herein are used to promote

wound healing (e.g., for a cut), treat an injury (e.g., broken bones, bleeding out, gun shot

injury, and/or reduce scarring during surgery). In some embodiments, surgery includes

cesarean. In some embodiments, an inducing agent and/or a chemical agent capable of

modulating activity of the inducing agent is also used.

WO wo 2020/069373 PCT/US2019/053545

[00343] In some embodiments, any of the nucleic acids (e.g., engineered nucleic acids)

OCT4, KLF4, and/or SOX2, antibodies activating (e.g., inducing expression of) a

vaccinia virus, retrovirus, herpes virus, or AAV) described herein are useful in healing an

injury and/or inflammation. In some embodiments, an inducing agent and/or a chemical

agent capable of modulating activity of the inducing agent is also used. In some

embodiments, the inflammation is hyperinflammation, which may be a side effect of aging.

In some embodiments, the hyperinflammation is inflammaging.

In some

[00344] In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

capable of inducing OCT4, KLF4, and/or SOX2 expression (e.g., expression vectors),

vaccinia virus, retrovirus, herpes virus, or AAV) described herein provide a healing capacity.

[00345] In some In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

(e.g., expression vectors) capable of inducing expression of OCT4, KLF4, SOX2, or a

combination thereof, engineered cells, engineered proteins, chemical agents activating (e.g.,

herpes virus, or AAV) described herein provide a healing capacity.

[00346] In some In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

(e.g., inducing expression of) OCT4, KLF4, SOX2, or a combination thereof, and/or recombinant viruses (e.g., lentivirus, adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) described herein are useful in enhancing or rejuvenating optimal or sub-optimal organs. As a non-limiting example, any of the compositions described herein

(e.g., recombinant viruses including recombinant AAV viruses) encoding OCT4, KLF4,

SOX2, or a combination thereof may be useful in enhancing or rejuvenating suboptimal

organs (e.g., from older individuals) that are used for transplantation or to promote organ

survival during transport or to promote organ survival after reimplantation of the organ into a

subject.

[00347] Any Any of of thethe nucleicacids nucleic acids (e.g., (e.g., engineered engineerednucleic acids) nucleic (e.g., acids) expression (e.g., vectors) expression vectors)

capable of inducing expression of OCT4, KLF4, SOX2, or a combination thereof, engineered

cells, engineered proteins, chemical agents activating (e.g., inducing expression of) OCT4,

KLF4, SOX2, or a combination thereof, antibodies activating (e.g., inducing expression of)

OCT4, KLF4, SOX2, or a combination thereof, and/or recombinant viruses (e.g., lentivirus,

adenovirus, alphavirus, vaccinia virus, retrovirus, herpes virus, or AAV) described herein

may be used to rejuvenate or increase the survival and longevity of cells (e.g., hematopoietic

stem cells, T-cells, etc.) that are used for transplantation. In some embodiments, recombinant

viruses (e.g., AAV viruses) encoding OCT4, KLF4, SOX2, or a combination thereof are

useful in rejuvenating or increasing the survival and longevity of cells (e.g., hematopoietic

stem cells, T-cells, etc.) that are used for transplantation.

In some

[00348] In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

herpes virus, or AAV) described herein is used to prevent or relieve the side effects of a toxin

and/or a drug (e.g., a chemotherapy) in a subject. Non-limiting examples of side effects

include hair loss and peripheral neuropathy. Chemotherapies include vincristine (VCS). See,

e.g., example 15. In certain embodiments, a composition comprising a recombinant virus

(e.g., AAV virus) encoding SOX2, KLF4, OCT4, or a combination thereof, is administered to

treat (e.g., recover from) or prevent the side effects induced by a toxin and/or damaging drug

therapy (e.g., a chemotherapy drug including VCS).

[00349] In some embodiments, any of the nucleic acids (e.g., engineered nucleic acids)

WO wo 2020/069373 PCT/US2019/053545

combination thereofengineered combination thereof, engineered cells, cells, engineered engineered proteins, proteins, chemicalchemical agents activating agents activating (e.g., (e.g.,

herpes virus, or AAV) described herein is administered to a subject to prevent or relieve the

side effects of a toxin and/or a drug (e.g., a chemotherapy).

In some

[00350] In some embodiments, embodiments, any any of the of the nucleic nucleic acids acids (e.g., (e.g., engineered engineered nucleic nucleic acids) acids)

retrovirus, herpes virus, or AAV) described herein is administered to a subject to protect a

tissue, organ, and/or entire body of the subject from radiation (e.g., prevent the damaging

effects of radiation). In certain embodiments, AAV encoding OCT4, SOX2, KLF4, or any

combination thereof, is administered to a subject to protect a tissue, organ, and/or entire body

of the subject from radiation protect (e.g., prevent the damaging effects of radiation).

[00351] Methods for identifying subjects suspected of having a condition may include

physical examination, subject's family medical history, subject's medical history, biopsy,

genetic testing, DNA sequencing of pathogens or the microbiome, proteomics, or a number of

imaging technologies such as ultrasonography, computed tomography, magnetic resonance

imaging, magnetic resonance spectroscopy, or positron emission tomography.

[00352] Effective amounts of the engineered nucleic acids (e.g., expression vectors,

including viral vectors), viruses (e.g., lentiviruses, retroviruses, adenoviruses, retroviruses,

alphaviruses, vaccinia viruses, or AAVs) or compositions thereof vary, as recognized by

those skilled in the art, depending on route of administration, excipient usage, and co-usage

with other active agents. The quantity to be administered depends on the subject to be

treated, including, for example, the age of the subject, the gravity of the condition, the weight

of the subject, the genetics of the subject, the cells, tissue, or organ to be targeted, or any

combination thereof.

Expression

[00353] Expression of of oneorormore one more transcription transcription factors of of factors the the present disclosure present (e.g., (e.g., disclosure

OCT4; KLF4; SOX2; or any combination thereof) may result in reprogramming of a cell,

tissue repair, tissue regeneration, increase blood flow, organ regeneration, improved

immunity, reversal of aging, counter senescence, or any combination thereof. Cellular

WO wo 2020/069373 PCT/US2019/053545

reprogramming may be determined by determining the extent of differentiation of a cell (e.g.,

by determining the expression of one or more lineage markers or pluripotency markers,

including OCT4, KLF4, SOX2, NANOG, ESRRB, NR4A2, and C/EBPa). The C/EBP). The

differentiation potential of a cell may also be determined using routine differentiation assays

or gene expression patterns. Tissue repair may be determined by tissue replacement and

tissue regeneration assays. For example, tissue replacement assays include wound healing

assays in cell culture or in mice. Tissue regeneration may be determined by quantifying a

particular cell type following expression of one or more transcription factors compared to

before expression of OCT4, KLF4, and SOX2 (see, e.g., the Examples provided below).

Tissue regeneration may be determined by quantifying a particular cell type following

expression of one or more transcription factors compared to before expression of OCT4;

KLF4; SOX2; or any combination thereof. In some instances, the methods described herein

promote organ regeneration (e.g. liver regeneration or reversal of liver fibrosis and regrowth).

In some instances, the methods described herein promote tissue and cell survival. Cell

survival in the face of adversity and damage may be determined using assays for cell viability

that are standard in the art (e.g., testing neuronal survival with the nano-glo live cell assay

from Promega corp.). In some instances, the methods described herein may prevent axonal or

Wallerian degeneration, which may be determined by quantifying the rate of axonal

degeneration after nerve crush in vitro using nerve cell cultures or in rat and mouse nerve

crush models known to those skilled in the art.

[00354] In some In some embodiments, embodiments, the the methods methods described described herein herein do not do not induce induce teratoma teratoma

formation. formation. In In some some embodiments, embodiments, expression expression of of OCT4, OCT4, SOX2, SOX2, KLF4, KLF4, or or aa combination combination

thereof or activation of OCT4, SOX2, KLF4, or a combination thereof in a subject, tissue, or

organ, results in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least

60%, at least 70%, at least 80%, at least 90%, or at least 100% reduction in teratoma

formation as compared to expression of OCT4, SOX2, KLF4, or a combination thereof and C- c-

MYC or activation of OCT4, SOX2, KLF4, or a combination thereof and c-MYC in the

subject, tissue, or organ. In some embodiments, expression of OCT4, SOX2, and KLF4 or

activation of OCT4, SOX2, and KLF4 in a subject, tissue, or organ, results in at least 10%, at

least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at

least 90%, or at least 100% reduction in teratoma formation as compared to expression of

OCT4, SOX2, and KLF4, and c-MYC or activation of OCT4, SOX2, KLF4, and c-MYC in

the subject, tissue, or organ. In some embodiments, the number of teratomas or the size of a

teratoma in a subject, tissue, or organ is the same or is reduced following expression of

WO wo 2020/069373 PCT/US2019/053545

OCT4, SOX2, KLF4, or a combination thereof or activation of OCT4, SOX2, KLF4, or a

combination thereof in a subject, tissue, or organ as compared to the number of teratomas or

the size of a teratoma in the subject, tissue, or organ prior to activation or expression of

OCT4, SOX2, KLF4, or a combination thereof.

[00355] In some embodiments, In some the the embodiments, methods described methods herein described do not herein induce do not unwanted induce cellcell unwanted

proliferation. In some embodiments, the unwanted cell proliferation is aberrant cell

proliferation, which may be benign or cancerous. In some embodiments, expression of

combination thereof in a subject, tissue, or organ reduces unwanted cell proliferation in a

subject, tissue, or organ, by at least 10%, at least 20%, at least 30%, at least 40%, at least

50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to

the same method with c-Myc expression or activation. In some embodiments, unwanted cell

proliferation in a subject, tissue, or organ is the same or is reduced following expression of

combination thereof in a subject, tissue, or organ as compared to the amount of unwanted cell

proliferation in the subject, tissue, or organ prior to activation or expression of OCT4, SOX2,

KLF4, or a combination thereof.

In some

[00356] In some embodiments, embodiments, the the methods methods described described herein herein do not do not induce induce tumor tumor

formation or tumor growth. In some embodiments, expression of OCT4, SOX2, KLF4, or a

combination thereof or activation of OCT4, SOX2, KLF4, or a combination thereof in a

subject, tissue, or organ reduces the number of tumors or the size of a tumor in a subject,

tissue, or organ, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at

least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to the same

method with c-Myc expression or activation. In some embodiments, the number of tumors or

the size of a tumor in a subject, tissue, or organ is the same or is reduced following

expression of OCT4, SOX2, KLF4, or a combination thereof or activation of OCT4, SOX2,

KLF4, or a combination thereof in a subject, tissue, or organ as compared to the number of

tumors or the size of a tumor in the subject, tissue, or organ prior to activation or expression

of OCT4, SOX2, KLF4, or a combination thereof. In some embodiments, a method

described herein does not induce cancer. In some embodiments, a method described herein

does not induce glaucoma.

Methods

[00357] Methods of reprogramming of reprogramming are are alsoalso provided provided herein. herein. In some In some embodiments, embodiments, a a

method of reprogramming described herein comprises reversing or rejuvenating the

epigenetic clock of a cell, tissue, organ, or a subject. In some embodiments, the epigenetic

WO wo 2020/069373 PCT/US2019/053545

clock may be partially or fully reversed. In some embodiments, the epigenetic clock of a cell,

tissue, organ, or a subject is measured using DNA methylation-based age (DNAmAGE or

DNAm age). In some embodiments, a method described herein reduces the DNAmAge age

of a cell by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

In some

[00358] In some embodiments, embodiments, a method a method of reprogramming of reprogramming described described herein herein comprises comprises

altering the expression of one or more genes associated with ageing. In some embodiments,

expression of a gene is increased by at least 1%, at least 5%, at least 10%, at least 20%, at

least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or

at least 100%. In some embodiments, expression of a gene is reduced by at least 1%, at least

5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least

70%, at least 80%, at least 90%, or at least 100%. In some embodiments, expression of one

or more genes following performance of a method is determined relative to expression of the

one or more genes prior to performance of the method. In some embodiments, expression of

one or more genes is determined relative to expression of the one or more genes in a young

cell, a young subject, a young tissue, a young organ, or any combination thereof. In some

embodiments, expression of one or more genes is determined relative to expression of the one

or more genes in an old cell, an old subject, an old tissue, an old organ, or any combination

thereof.

[00359] A gene associated with ageing may be a gene whose expression is altered in an

old, an old tissue, an old organ, an old subject, or any combination thereof as compared to a

young counterpart. In some embodiments, the gene associated with ageing is

1700031P21Rik, 1810053B23Rik, 2900045O20Rik, 2900060B14Rik, 4921504E06Rik,

4930402F11Rik, 4930453C13Rik, 4930455B14Rik, 4930500H12Rik, 4930549P19Rik,

4930555B11Rik, 4930556J02Rik, 4932442E05Rik, 4933431K23Rik, 4933438K21Rik,

6720475M21Rik, 9830132P13Rik, A430010J10Rik, A530064D06Rik, A530065N20Rik,

Gm10248, Gm10513, Gm10635, Gm10638, Gm10718, Gm10722, Gm10800, Gm10801,

Gm11228, Gm11251, Gm11264, Gm11337, Gm11368, Gm11485, Gm11693, Gm12793,

Gm13050, Gm13066, Gm13323, Gm13339, Gm13346, Gm13857, Gm14387, Gm14770,

Gm15638, Gm16072, Gm16161, Gm16181, Gm17200, Gm17791, Gm18025, Gm18757,

Gm18795, Gm18848, Gm19719, Gm20121, Gm20356, Gm2093, Gm21738, Gm21940,

Gm22933, Gm24000, Gm24119, Gm25394, Gm26555, Gm27047, Gm28262, Gm28530,

Gm29295, Gm29825, Gm29844, Gm3081, Gm32051, Gm32122, Gm33056, Gm33680,

Gm34354, Gm34643, Gm3551, Gm36660, Gm36948, Gm37052, Gm37142, Gm37262,

Gm37535, Gm37569, Gm37589, Gm37647, Gm37648, Gm37762, Gm38058, Gm38069,

Gm38137, Gm38218, Gm39139, Gm42535, Gm42680, Gm42895, Gm42994, Gm43027,

Gm43158, Gm43288, Gm43366, Gm44044, Gm44081, Gm44187, Gm44280, Gm44535,

Gm45338, Gm45644, Gm45740, Gm46555, Gm46565, Gm4742, Gm47485, Gm47853,

Gm47992, Gm48225, Gm48314, Gm48383, Gm48673, Gm48804, Gm48832, Gm4994,

Serpinb9b, Skint1, Skint3, Skint5, Slc10a5, Slc6a4, Smok2a, Tcaf3, Tomm201, Trcgl, Trcg1, Trdn,

Ugtla6a, Ugt1a6a, Usp171a, Usp17la, Vmn1r178, Vmn1r179, Vmn1r33, Vmn1r74, Vmn1r87, Vmn2r102,

Vmn2r113, Vmn2r17, Vmn2r52, Vmn2r66, Vmn2r68, Vmn2r76, Vmn2r78, Wnt16,

0610040J01Rik, 1700080N15Rik, 2900064F13Rik, 4833417C18Rik, 4921522P10Rik,

4930447C04Rik, 4930488N15Rik, Ace, Ackr1, Acot10, Acvrl, Acvr1, Adamts17, Adra1b, Adralb,

Dgkg, Dlk2, Dnajal-ps, Dnaja1-ps, Drd2, Dsel, Dytn, Ecscr, Edn1, Ednrb, Efemp1, Elfn2, Ephal0, Epha10,

Gfra2, Ggtl, Ggt1, Gm10416, Gm14964, Gm17634, Gm2065, Gm32352, Gm33172, Gm34280,

Gm35853, Gm36298, Gm36356, Gm36937, Gm3898, Gm42303, Gm42484, Gm42537,

Gm42743, Gm43151, Gm43843, Gm44545, Gm44722, Gm45516, Gm45532, Gm47494,

Kbtbd12, Kcnj11, Kcnk4, Kdelc2, Klhl33, Lame3, Lamc3, Lilra5, Lman11, Lrfn2, Lrrc38, Lrrn4cl,

Tmem132d, Tmem200a, Tmem44, Trpc4, Trpv4, Unc5b, Vgf, Vmn1r90, Vwc21, Wfikkn2,

the gene is a sensory gene.

[00360] In some embodiments, In some a method embodiments, described a method herein described reduces herein expression reduces of of expression

0610040J01Rik, 1700080N15Rik, 2900064F13Rik, 4833417C18Rik, 4921522P10Rik,

4930447C04Rik, 4930488N15Rik, Ace, Ackrl, Ackr1, Acot10, Acvrl, Acvr1, Adamts17, Adralb,

Gm35853, Gm36298, Gm36356, Gm36937, Gm3898, Gm42303, Gm42484, Gm42537,

Gm42743, Gm43151, Gm43843, Gm44545, Gm44722, Gm45516, Gm45532, Gm47494,

Tmem132d, Tmem200a, Tmem44, Trpc4, Trpv4, Unc5b, Vgf, Vmn1r90, Vwc21, Wfikkn2,

Wnt11, Wnt6, Zeb2os, Zfp608, Zfp976, or any combination thereof. See, e.g., Table 5 for

genes associated with ageing.

[00361] In some embodiments, a method described herein increases expression of

1700031P21Rik, 1810053B23Rik, 2900045O20Rik, 2900060B14Rik, 4921504E06Rik,

4930402F11Rik, 4930453C13Rik, 4930455B14Rik, 4930500H12Rik, 4930549P19Rik,

4930555B11Rik, 4930556J02Rik, 4932442E05Rik, 4933431K23Rik, 4933438K21Rik,

6720475M21Rik, 9830132P13Rik, A430010J10Rik, A530064D06Rik, A530065N20Rik,

WO wo 2020/069373 PCT/US2019/053545

Gm10248, Gm10513, Gm10635, Gm10638, Gm10718, Gm10722, Gm10800, Gm10801,

Gm11228, Gm11251, Gm11264, Gm11337, Gm11368, Gm11485, Gm11693, Gm12793,

Gm13050, Gm13066, Gm13323, Gm13339, Gm13346, Gm13857, Gm14387, Gm14770,

Gm15638, Gm16072, Gm16161, Gm16181, Gm17200, Gm17791, Gm18025, Gm18757,

Gm18795, Gm18848, Gm19719, Gm20121, Gm20356, Gm2093, Gm21738, Gm21940,

Gm22933, Gm24000, Gm24119, Gm25394, Gm26555, Gm27047, Gm28262, Gm28530,

Gm29295, Gm29825, Gm29844, Gm3081, Gm32051, Gm32122, Gm33056, Gm33680,

Gm34354, Gm34643, Gm3551, Gm36660, Gm36948, Gm37052, Gm37142, Gm37262,

Gm37535, Gm37569, Gm37589, Gm37647, Gm37648, Gm37762, Gm38058, Gm38069,

Gm38137, Gm38218, Gm39139, Gm42535, Gm42680, Gm42895, Gm42994, Gm43027,

Gm43158, Gm43288, Gm43366, Gm44044, Gm44081, Gm44187, Gm44280, Gm44535,

Gm45338, Gm45644, Gm45740, Gm46555, Gm46565, Gm4742, Gm47485, Gm47853,

Gm47992, Gm48225, Gm48314, Gm48383, Gm48673, Gm48804, Gm48832, Gm4994,

Msh4, Mucl2, Mug1, Mybl2, Myh15, Nek10, Neurod6, Nr1h5, Olfr1042, Olfr1043,

Vmn2r113, Vmn2r17, Vmn2r52, Vmn2r66, Vmn2r68, Vmn2r76, Vmn2r78, Wnt16, or any

combination thereof.

Aspects

[00362] Aspects of the of the present present disclosure disclosure relate relate to methods to methods comprising comprising resetting resetting the the

thereof in vitro. Aspects of the present disclosure relate to methods comprising resetting the

combination thereof in vivo. In some embodiments, resetting the transcriptional profile an

old cell, an old organ, an old tissue, an old subject and/or any combination thereof comprises

WO wo 2020/069373 PCT/US2019/053545

altering the gene expression of one or more genes associated with ageing. In some

embodiments, resetting the transcriptional profile an old cell, an old organ, an old tissue, an

old subject and/or any combination thereof comprises reversing the epigenetic clock. In

some embodiments, the transcription profile of an old cell is reset. In some embodiments, the

transcriptional profile of an old cell, an old organ, an old tissue, an old subject, or any

combination thereof is reset to that of a young cell, a young tissue, a young organ, a young

subject, or any combination thereof. In some embodiments, a method described herein

reverses one or more changes in gene expression that are detected between an old cell, an old

organ, an old tissue, an old subject, or any combination thereof and a control. In some

embodiments, the control is a young cell, a young organ, a young tissue, a young subject, or

any combination thereof. In some embodiments, the transcriptional profile of an old cell, an

old organ, an old tissue, an old subject, or any combination thereof is changed from a young

counterpart. In some embodiments, a method described herein resets at least 10%, at least

90%, or 100% of the gene expression change in an old cell, an old organ, an old tissue, an old

subject, or any combination thereof to a young level. In some embodiments, a sensory gene is

a sensory receptor gene. Without being bound by a particular theory, resetting of a sensor

receptor gene expression level in an aged cell to a young level may be indicative of an

improvement of retina ganglion cell function.

[00363] In some In some aspects, aspects, the the cellular cellular reprogramming reprogramming methods methods described described herein herein may may be be

used to promote the transdifferentiation of cells, which may be useful in treatment of disease.

In some embodiments, the methods described herein may improve the efficieny of existing

methods of transdifferentiation. For example, OCT4, SOX2, KLF4, or a combination thereof

may be activated (e.g., expressed) in one cell type along with one or more perturbations of

genes that affect cell fate to promote lineage reprogramming or conversion to another cell

type. In some embodiments, the perturbation is reducing expression of a lineage determining

factor. In some embodiments, the perturbation is expression of a lineage determining factor.

In some embodiments, the lineage determining factor is a lineage transcription factor.

[00364] As aAsnon-limiting a non-limiting example, example, night night blindness blindness is caused is caused by rod by rod death death and and daytime daytime

blindness is caused by cone death. Cell types including cones, rods, and muller cells could be

reprogrammed into another cell type needed to restore vision. For example, loss of Nrl

promotes transdifferentiation of adult rods into cone cells. See, e.g., Montana et al., Proc

Natl Acad Sci USA. 2013 U S A. Jan 2013 29;110(5):1732-7. Jan InIn 29;110(5):1732-7. some embodiments, some transcription embodiments, factors transcription factors

that promote rod cell fate include Otx2, Crx and Nrl. As a non-limiting example, Müller glia

WO wo 2020/069373 PCT/US2019/053545

(MG) can be reprogrammed into rod cells by expressing B-catenin, Otx2, Crx, -catenin, Otx2, Crx, and and Nrl. Nrl. See, See,

e.g., Yao et al., Nature. 2018 Aug;560(7719):484-488.

As another

[00365] As another non-limiting non-limiting example, example, pancreatic pancreatic alpha alpha may may be reprogrammed be reprogrammed intointo

beta cells for treating autoimmune diseases and diabetes. Transcription factors including

Pdx1 and MafA Pdx and MafA can can be be used used to to reprogram reprogram mouse mouse alpha alpha cells cells into into beta beta cells. cells. See, See, e.g., e.g., Xiao Xiao et et

al., Cell Stem Cell. 2018 Jan 4;22(1):78-90.e4.

Additional

[00366] Additional non-limiting examples non-limiting examples of of transdifferentiation transdifferentiationinducing factors inducing for factors for

production of various cell types may be found in Cieslar-Pobuda et al., Cielar-Pobuda et al., Biochim Biochim Biophys Biophys

Acta Mol Cell Res. 2017 Jul;1864(7):1359-1369, which is herein incorporated by reference in

its entirety. See e.g., Table 4 of Cieslar-Pobuda etal., Cielar-Pobuda et al.,Biochim BiochimBiophys BiophysActa ActaMol MolCell CellRes. Res.

2017 Jul;1864(7):1359-1369.

[00367] Induction Induction of OCT4, of OCT4, SOX2, SOX2, KLF4, KLF4, or aorcombination a combination thereof thereof may may increase increase the the

efficiency of trandifferentiation of cells by at least 1%, at least 5%, at least 10%, at least 20%,

at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%,

at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at

least 700%, at least 800%, at least 900%, or at least 1000%, including all values in between,

as compared to a control. The efficiency of transdifferentiation may be measured by any

suitable method including comparing the percentage of cells that were transdifferentiated

when OCT4, SOX2, KLF4, or a combination thereof was activated as compared to control

cells in which OCT4, SOX2, KLF4, or a combination thereof was not activated.

These

[00368] These andand other other aspects of aspects of the the present presentinvention inventionwill be further will appreciated be further upon appreciated upon

consideration of the following Examples, which are intended to illustrate certain particular

embodiments of the invention but are not intended to limit its scope, as defined by the claims.

EXAMPLES

[00369] In order In order thatthat the the present present disclosure disclosure may may be more be more fully fully understood, understood, the the following following

examples are set forth. The synthetic and biological examples described in this application

are offered to illustrate the compounds, pharmaceutical compositions, and methods provided

herein and are not to be construed in any way as limiting their scope.

Example 1: Development of an adenovirus-associated virus (AAV) vector for inducible

expression of OCT4, SOX2, and KLF4 (OSK) in mammalian cells.

[00370] An AAV vector that is capable of expressing OCT4, SOX2, and KLF4 in

mammalian cells was developed as described herein. As shown in FIG. 1, such a vector comprises a TRE3G promoter (SEQ ID NO: 7), nucleic acids (e.g., engineered nucleic acids) encoding OCT4, SOX2, KLF4, and an SV40 polyA (SV40pA) terminator sequence (SEQ ID

NO: 8). This vector will be referred to as TRE3G-OSK-SV40pA. Nucleic acid (e.g.,

engineered engineered nucleic nucleic acid) acid) sequences sequences encoding encoding self-cleaving self-cleaving peptides peptides (T2A, (T2A, aa 2A 2A peptide, peptide, SEQ SEQ

ID NO: 9) were used to separate the nucleic acids (e.g., engineered nucleic acids) encoding

OCT4, SOX2, and KLF4. As shown in FIG. 2, the entire vector is 7,408 base pairs in length

and two inverted terminal repeats (ITRs) flank the OSK sequences. The restriction enzyme

digestion sites in the vector are depicted in FIG. 3. A schematic mapping the features shown

in the vector maps of FIGS. 4A-4AL onto the nucleic acid (e.g., engineered nucleic acid)

sequence of the vector is shown in FIGS. 2-3. The restriction enzyme cut sites are shown in

Table 3 below. As shown in FIGS. 5A-5D, the open reading frame (ORF frame 3) encoding

OSK and intervening 2A peptides (T2A peptides) is 3,610 base pairs.

Table 3. Restriction Enzyme Cut Sites in the TRE3G-OSK-SV40pA vector. Enzymes Sites Location AatII 1 4033 11 Acc651 Acc65I 6074 Afel 11 5333 AfIII Aflll 11 2847 Alel AleI 1 5656 1 BbvCI 5098 Bcll 1* 4246* 11 Bmtl BmtI 3349 1 Bpu10I Bpu101 5098 BsaBI 1* 6098* 11 BspQI 1436 1 BsrDI 371 1 1 BstAPI 4016 BstXI 11 4667 BstZ17I 1 5078 1 1 EcoRI 1893 1 Kpnl KpnI 6078 1 Nhel 3345 NotI 1 2276 1 PaeR7I 4449 PfIFI PflFI 11 3546 RsrII Rsrll 1 3542 SacII 1 3765 11 Sapl SapI 1436 1 Scal 7358

143

SexAI 1* 2330* Spel 1 5907 1 1 Tth1111 Tth111I 3546 1 Xhol 4449 Zral 1 4031 4031

[00371] The The vector vector shown shown in FIGS. in FIGS. 3 and 3 and 4A-4AL 4A-4AL was was cloned cloned using using routine routine methods. methods.

Briefly, a TRE3G promoter sequence (SEQ ID NO: 7) from Clonetech was synthesized using

flanking restriction sites, primers were designed to clone OSK out of a TetO-FUW-OSKM

plasmid, and a stop codon was added. To make the vector shorter, a short SV40 sequence

was synthesized with flanking restriction cut sites. Whereas conventional AAV vectors

encoding OSK is over the packaging limit of AAV, could only be packaged into AAV9

capsid with low titer (less than 2 X 10^12 particles per viral prep), and the low titer virus is

not functional due to possible truncation as shown in FIG.17. The vector depicted in FIGS. 3

and 4A-4AL produced virus with more than 2 X x 10^12 viral partial per prep or 1 X 10^13 per

mL (data not shown).

To determine

[00372] To determine whether whether the the OSK OSK vector vector could could be used be used for for inducible inducible OSK OSK

expression in mammalian cells, the OSK vector and was packaged into different serotypes of

AAV virus (AAV9 (FIG. 6A), AAV2 (FIG. 6B), and AAV. PhP.b(FIG. AAV.PhP.b (FIG.6C)) 6C))using usingroutine routine

methods. Additional batches of AAV9 and AAV.PHP.b virus with a vector encoding rtTA3

(Tet-On system) and AAV2 virus with a vector encoding tTA (Tet-Off system) were

produced. Then, mammalian 293T cells were co-infected with the OSK virus along with the

same serotype of rtTA3 or tTA virus. Cells were subsequently treated with or without

doxycycline (DOX) and expression of OCT4, KLF4 and the loading control H3 was

determined by western blot with antibodies against OCT4, KLF4, and H3.

As shown

[00373] As shown withwith the the Tet-On Tet-On system system in FIG. in FIG. 6A, 6A, doxycycline doxycycline treatment treatment increased increased

OCT4 and KLF4 expression in 293T cells infected with AAV9 viruses encoding OSK and

rtTA3. The OSK expression could also be controlled with a Tet-Off system. DOX treatment

decreased OCT4 and KLF4 expression in 293T cells infected with OSK AAV2 and AAV2

with a vector driving tTA expression under a constitutive CAG promoter (FIG. 6B).

Furthermore, OSK expression could be tightly controlled even after stimulation of transgene

expression. As shown in the fourth lane of FIG. 6C, one day of DOX treatment is sufficient

to increase OCT4 and KLF4 expression in 293T cells infected with TRE3G-OSK-SV40pA

AAV.PHP.b virus and with Ubc-rTtA3-p2a-mkate AAV.PHP.b virus. Removal of DOX for

three days after one day of DOX treatment, however, returns OCT4 and KLF4 expression

WO wo 2020/069373 PCT/US2019/053545

back to uninduced levels (last lane of FIG. 6C). The Ubc-rtTA3-p2a-mkate vector comprises

a constitutive Ubc promoter that drives expression of rtTA3, a self-cleaving 2A peptide, and a

far-red fluorescent protein (mKate).

[00374] Therefore, Therefore, an AAV an AAV vector vector thatthat allows allows for for controlled controlled expression expression of OSK of OSK in in

mammalian cells (e.g., in vivo) was developed. Furthermore, the AAV vector was packaged

into different AAV serotypes that successfully delivered a functional vector into 293T cells.

Example 2: AAV encoding OSK promoted optic nerve regeneration and survival of retina

ganglion cells (RGCs) nerves after nerve crush in an inducible manner.

[00375] To To

[00375] determinewhether determine whether OSK OSK could couldbebedelivered by by delivered AAV AAV and and inducibly expressed inducibly expressed

in vivo, AAV virus with the TRE-OSK-SV40 vector and AAV virus encoding tTA under the

CAG constitutive promoter were produced through routine methods and injected into the

retina of mice. Next, an optical coherence tomography (OCT) section was stained with

antibody against RBPMS to identify retina ganglion cells (RGCs) and with an antibody

against KLF4 to detect KLF4 expression. As shown in FIG. 7A, KLF4 was expressed in

RGCs (RBPMS-positive cells), which suggested that the vectors were functional.

[00376] The The inducibility inducibility of the of the system system was was alsoalso tested tested in vivo. in vivo. In the In the absence absence of DOX of DOX

treatment, OCT4 and KLF4 were expressed in the mouse retina as determined by whole

retina mount staining (FIG. 7B, top). After four days of DOX treatment, however, OCT4 and

KLF4 staining was significantly reduced, indicating that expression from the TRE-OSK-

SV40 vector was turned off (FIG. 7B, bottom). Therefore, OSK vector expression could be

tightly controlled.

[00377] To determine To determine whether whether inducible inducible OSK OSK expression expression could could induce induce partial partial

reprogramming and promote regeneration following nerve damage, AAV virus with the TRE-

OSK-SV40 vector and AAV virus encoding tTA under the CAG constitutive promoter were

injected into the retina of 4-week old mice (n=6) as shown in the experimental timeline

provided in FIG. 7C. As a control, a separate cohort of mice (n=2) were only injected with

the OSK virus. Mechanical damage was induced through optic nerve crush in both cohorts

two weeks after virus injection. To trace axon regeneration by fluorescent microscopy of the

optic optic nerve, nerve,fluorescently labeled fluorescently cholera labeled toxin B-subunit cholera (CTB) was toxin -subunit intraocularly (CTB) injected was intraocularly injected

into into mice mice and and perfusion perfusion was was performed performed two two days days after after CTB CTB injection. injection. Axon Axon regeneration regeneration and and

axon survival analysis was subsequently conducted.

[00378] AxonAxon regeneration regeneration was was determined determined by estimating by estimating the the number number of axons of axons per per nerve. nerve.

As shown in FIG. 7D, co-administration of OSK and tTA virus significantly promoted optic nerve regeneration away from the site of the optic nerve crush compared to administration of

OSK virus alone. This effect was also visually apparent when comparing the fluorescence

intensity of optic nerves from mice receiving both OSK and tTA virus compared to mice

receiving OSK virus alone. The fluorescence intensity of optic nerves from mice receiving

both viruses was higher than that of mice receiving OSK virus alone, indicating that nerve

regeneration was higher with combination treatment (FIGS. 7E-7F).

[00379] To show To show thatthat the the observed observed axonaxon regeneration regeneration after after crush crush injury injury was was specifically specifically

mediated by OSK, an axon regeneration experiment was used to compare the effects of tTA

virus in combination with (1) TRE-OSK virus with no DOX treatment, (2) TRE-d2EGFP

virus with no DOX treatment, and (3) TRE-OSK virus with DOX treatment. The

experimental timeline of treatments (1)-(3) are indicated in FIGS. 8B, 8D, and 8F,

respectively. Fluorescently-labeled CTB was used to visualize axons. As shown in FIG. 8A

and 8G, the extent of optic nerve regeneration in mice in which OSK expression was induced

(mice receiving OSK and tTA viruses in the absence of DOX) was very significant at 200 um µm

and 500 um µm from crush site. In contrast, even when d2EGFP expression was induced (mice

receiving d2EGFP and tTA viruses in the absence of DOX), minimal regeneration was

observed (FIG. 8A and FIG. 8C). Notably, axon regeneration was dependent on induction of

OSK expression. When mice were treated with DOX to inhibit OSK expression as outlined

in FIG. 8D, administration of tTA and OSK viruses did not induce axon regeneration (FIG.

8A). The intensity of CTB-labeled axons in these DOX-treated mice were similar to mice

receiving control d2EGFP virus (compare FIG. 8E with FIG. 8C). Therefore, administration

of an AAV-based inducible OSK expression system could be used to promote regeneration

following optic nerve damage.

[00380] The The effect effect of OSK of OSK on the on the survival survival raterate of retina of retina ganglion ganglion cells cells (RGCs) (RGCs) was was alsoalso

assessed. As shown in FIGS. 9A-9D, OSK significantly increases RGC survival rate. RGCs

(RBPMS positive cells) that were infected with OSK and tTA virus (green) or uninfected

with both (red) shown following optic nerve crush, OSK-infected RGC had 3 times higher

survival rate (54% vs 18%) after crush compared to cells without OSK infection,

quantification from a series of pictures like shown (FIG. 9A). Therefore, the percentage of

RBPMS-positive cells expressing KLF4 (OSK-infected cells) was lower than 40% before

crush, but significantly increased to around 70% following optic nerve crush due to its higher

survival rate. While the percentage of d2EGFP-infected cells maintained at 35-40% after

crush. This indicates a strong cell protection effect from OSK expression (FIG. 9B). As

shown in FIG. 9C, in d2EGFP or OSK plus CAG-tA CAG-tTA(SEQ (SEQID IDNO: NO:32) 32)AAV AAVinfected infected

WO wo 2020/069373 PCT/US2019/053545

retina, there is no significant difference in RGC number (RBPMs positive) without

uncrushed, but after crush there is clearly more RGCs survived when they infected with OSK

and CAG-tA CAG-tTAcompared comparedto tothose thoseinfected infectedwith withd2EGFP d2EGFPand andCAG-tTA. CAG-tTA.FIG. FIG.9D 9Dshows showsthe the

quantification of survived RGC numbers from each group. Though lower than 40% cells

infected with both OSK and CAG-tA CAG-tTAAAV, AAV,it itincreases increasesthe thetotal totalsurvival survivalRGC RGCnumber number

compared to d2EGFP(542 compared to 323).

[00381] mTOR activation has reported as a pathway for optic nerve regeneration (Parker

et al., Science, 322(5903), 963-966 Nov. 2008). To determine whether OSK expression

activated the mTOR pathway, control and OSK virus-infected cells were imaged using

antibodies against RBPMS and phosphorylated S6 (pS6) in the absence of damage

(uncrushed) and after damage (crushed). Representative images of the staining is shown in

FIG. 10A, and as quantified in FIG. 10B, the percentage of pS6-positive cells was not

significantly different between control cells and OSK-infected cells following optic nerve

crush.

Example 3. An AAV Tet-On system induces faster gene expression compared to an AAV Tet-

Off system in retinal cells after nerve crush.

[00382] To compare To compare the the raterate of gene of gene expression expression between between AAV-based AAV-based Tet-On Tet-On and and Tet-Off Tet-Off

systems, TRE-d2EGFP virus and (1) virus encoding tTA (Tet-Off) or (2) virus encoding rtTA

(Tet-On) were administered into the retina of 4-week old mice. In the Tet-Off system, mice

were given DOX starting from virus injection and DOX was removed for 3 days, 5 days or 8

days (FIG. 11A). As a control, a cohort of mice in the Tet-Off system were given no DOX.

Approximately 8 days of DOX removal was needed to induce the same level of GFP

expression as no DOX treatment in the Tet-Off system (FIG. 11B). In the Tet-On system,

mice were treated as indicated in FIG. 11C. GFP expression was observed after only 2 days

of DOX treatment in the Tet-On system (FIG. 11D). Therefore, a shorter period of time was

needed to induce transgene expression in mice retina infected with an AAV-based Tet-On

system compared to infection with an AAV-based Tet-Off system.

Example 4: An AAV vector encoding mutant reverse tetracycline transactivator (rtTA)

showed low leakiness in the liver of mice and low toxicity.

[00383]

[00383] As As shown shown in in FIGS. FIGS. 13A-13C, 13A-13C, OCT4, OCT4, SOX2, SOX2, andand KLF4 KLF4 through through AAV9 AAV9 delivery delivery

(TRE-OSK with UBC-rtTA4) can be successfully induced in liver of the mice with DOX

treatment, shown with both western blot and immune staining. While mice with transgene of

WO wo 2020/069373 PCT/US2019/053545

OCT4, SOX2, KLF4 died after 2 days-induction from doxycycline water (FIG. 14) due to

generalized cytological and architectural dysplasia in the intestinal epithelium, expression

from the OCT4, SOX2, and KLF4 AAV described herein did not cause toxicity or teratoma

in vivo even with continuous expression through doxycycline administration in their drinking

water. No teratoma or body weight loss were detected for three months when AAV9

encoding these three transcription factors were delivered to the entire body of mice (FIG. 14).

Example 5: Expression of OCT4, SOX2, and KLF4 induced partial reprogramming in mice.

FIGS.

[00384] FIGS. 15A-15B 15A-15B showshow thatthat the the expression expression of histone of histone and and ChafChaf (Chromatin (Chromatin

assembly factor) genes declined during aging in ear fibroblasts from aged mice (12 months or

15 months) compared to those from young mice, short term of OSKM (3 days) or OSK

expression (5 days) induction reset their gene expression level to young state, without making

them into stem cell (e.g., Nanog was not been turned on).

[00385] Conventional AAV vectors encoding OSK is over the packaging limit of AAV

(e.g., 4.7 Kb), could only be packaged into AAV9 capsid with low titer (less than X 2 10^12 X 10^12

particles per viral prep), and the low titer virus is not functional (e.g., no overexpression of

OCT4 or KLF4 was detected) due to possible truncation as shown in FIG. 16.

Example 6: An AAV vector encoding mutant reverse tetracycline transactivator (rtTA)

showed low leakiness in the liver of mice.

[00386] A Tet-On A Tet-On system system comprising comprising rtTA4 rtTA4 (SEQ(SEQ ID NO: ID NO: 13) 13) was was alsoalso tested tested in vivo in vivo

using recombinant using recombinant AAV9 AAV9 viruses. viruses. Two Two AAV vectors AAV vectors comprising comprising components components shown in shown FIG. in FIG.

13B were used. AAV virus encoding rtTA4 operably linked to a UBC promoter (pAAV-

UBC-rtTA4-WPRE3-SV40pA vector is provided as SEQ ID NO: 17 and an exemplary vector

map of SEQ ID NO: 17 is provided in FIG. 12) and AAV virus encoding an AAV TRE3G-

OSK-SV40pA vector (SEQ ID NO: 16) with a vector map depicted in FIG. 3 were

administered to mice. Mice were treated without doxycycline or with doxycycline and liver

samples were collected. As shown in the immunofluorescence images of FIG. 13A, in the

absence of doxycycline, KLF4 expression was not detectable in the liver. When mice were

treated with doxycycline through their drinking water, KLF4 expression was detected in the

liver (FIG. 13A). These results were also evident by western blot using antibodies against

OCT4, KLF4, and SOX2 to determine expression of these protein (FIG. 13C). Actin was

used as a loading control (FIG. 13C). OCT4, KLF4, and SOX2 were only detected in the

liver when mice were treated with doxycycline (FIG. 13C).

Example 7. Modified mRNAs encoding OCT4, SOX2, and KLF4(OSK) induced expression of

OSK in mouse fibroblasts.

Mouse

[00387] Mouse fibroblasts fibroblasts werewere successfully successfully transfected transfected withwith modified modified mRNAmRNA encoding encoding

OCT4, OCT4, SOX2, SOX2,KLF4, KLF4,and c-MYC and (OSKM). c-MYC LipofectamineTM (OSKM). MessengerMAXTM Lipofectamine MessengerMAX Transfection Reagent from Invitrogen was used to transfect the modified mRNAs. The

modifications were complete substitution of either 5-methylcytidine (5mC) for cytidine or

pseudouridine (psi) for uridine. See, e.g., Warren et al., Cell Stem Cell. 2010 Nov 5;7(5):618-

30; Mandal et al., Nat Protoc. 2013 Mar; 8(3):568-82. The dose of each RNA that was used is

provided in Table 4 below. The numbers 1-5 in the first column of Table 4 correspond to the

numbers 1-5 in FIG. 17.

Table 4. Doses of mRNA administered.

mRNA (ug) (µg)

S Total NDG O o K M 11 0 0 0 0 0 0 0

2 0.2 0.6 0.2 0.2 0.2 1.4 1X

3 0.4 1.2 0.4 0.4 0.4 0.4 2.8 2.8 2X 2X

4 0.8 2.4 0.8 0.8 0.8 5.6 4X

5 1.2 3.6 3.6 1.2 1.2 1.2 8.4 8.4 6X

[00388] A western blot was used to confirm that administration of the modified mRNA

induced expression of protein in the mouse fibroblasts. As shown in FIG. 17, transfection of

OSK modified mRNA into mouse fibroblasts cells to induce expression of OCT4, KLF4, and

SOX2 protein (NDG and zsGreen are modified mRNA that express green fluorescent protein

to indicate the efficiency of transfection).

[00389] This

[00389] This example shows example shows that that delivery deliveryofofRNA (e.g., RNA mRNA, (e.g., modified mRNA, RNA, modified modified RNA, modified

mRNA, etc.) encoding OCT4, KLF4, and SOX2 to mouse cells is feasible. These findings

may be extended to in vivo delivery of mRNA encoding OCT4, KLF4, and SOX2. As an example, for in vivo muscle delivery, electroporation, is used. As an example, for liver and other internal organ delivery, nanoparticles comprising RNA encoding OCT4, KLF4, and

SOX2, nanoparticles are used. See, e.g., Dong et al., Nano Lett. 2016 Feb 10;16(2):842-8.

Example 8. Chemical reprogramming of cells.

A non-limiting

[00390] A non-limiting of aofprotocol a protocol to chemically to chemically reprogram reprogram a mouse a mouse embryonic embryonic

fibroblast to an induced pluripotent stem cell is provided below. A similar protocol may be

found at Zhao et al., Cell. 2015 Dec 17;163(7):1678-91. FIG. 21 shows the results after

using the protocol provided below.

[00391]

[00391] Stage 1 Stage 1

100 ng/ml bFGF

0.5 mM VPA,

20 M µMCHIR99021, CHIR99021,

10 µM M 616452, 616452,

5 µM M tranylcypromine, tranylcypromine,

50 µM M forskolin, forskolin,

0.05 µM 0.05 AM580 M AM580 5 µM EPZ004777 M EPZ004777 On day 12, the cells were trypsinized, harvested and then re-plated at 50,000-200,000 cells

per well of a 6-well plate (1:10-15)

During days 12-16, concentrations of bFGF, CHIR, and forskolin were reduced to 25 ng/ml,

10 uM, µM, and 10 uM, µM, respectively.

On day 16, XEN-like epithelial colonies were formed and the culture was changed into stage

2 medium

[00392] Stage 2

25 ng/ml bFGF,

0.5 mM VPA,

10 M µMCHIR99021, CHIR99021, 10 µM 616452, M 616452, 5 µM tranylcypromine, M tranylcypromine,

10 uM µM forskolin,

0.05 M µMAM580, AM580, 0.05 µM 0.05 DZNep, M DZNep, 0.5 µM 0.5 5-aza-dC, M 5-aza-dC,

5 µM SGC0946 M SGC0946

[00393] On day 28, the culture was transferred into stage 3 medium.

[00394] Stage 3

N2B27-2iL medium

3 M µMCHIR99021, CHIR99021, 1 µM PD0325901, M PD0325901, 1,000 U/ml LIF

After

[00395] After another another 8-128-12 days, days, 2i-competent, 2i-competent, ESC-like, ESC-like, and and GFP-positive GFP-positive (if (if using using

pOct4-GFP reporter) CiPSC colonies emerged and were then picked up for expansion and

characterization.

Example 9. Expression of OCT4, SOX2, and KLF4 improved axon regeneration in adult and

aged mice after optic nerve crush injury.

[00396] The The Tet-Off Tet-Off system system depicted depicted in FIG. in FIG. 22, 22, top top panel panel was was usedused to determine to determine whether whether

a vector encoding TRE-OSK-SV40 (SEQ ID NO: 16) could be used to promote optic nerve

axon regeneration in adult (3 month old) and aged (12 month old) mice.

[00397] AAV2

[00397] AAV2 virus virus with with thethe TRE-OSK-SV40 TRE-OSK-SV40 vector vector andand AAV2 AAV2 virus virus encoding encoding tTAtTA

under the CAG constitutive promoter were injected into the retina of 1 month old, 3 month

old, or 12 month old mice (n=5-9), similar to the experimental timeline provided in FIG. 7C.

As a control, a separate cohort of 1 month old mice (n=5-6) were injected with AAV2 virus

with a AAV2 vector TRE-d2EGFP-SV40 and the AAV2 virus encoding tTA. Mechanical

damage was induced through optic nerve crush in both cohorts two weeks after virus

injection. To trace axon regeneration by fluorescent microscopy of the optic nerve,

fluorescently labeled cholera toxin B-subunit ß-subunit (CTB) was intraocularly injected into mice two

weeks after optic nerve crush injury and perfusion was performed two days after CTB

injection. Axon regeneration analysis was subsequently conducted.

[00398] As shown in FIGS. 23A-23B, administration of AAV2 virus encoding OSK

increased the number of estimated axons per nerve in 1 month old (young), 3 month old

(adult), and 12 month old (aged) mice relative to administration of control virus encoding

d2EGFP. Furthermore, TRE-OSK virus also increased the survival of RGCs after optic nerve

injury in adult (3 month old) and aged (12 month old) mice compared to control GFP (FIG.

23C). Therefore, OSK expression surprisingly promoted axon regeneration and RGC

survival after nerve crush injury in young, adult, and aged mice.

WO wo 2020/069373 PCT/US2019/053545

[00399] Next, the impact of the length of time of OSK expression on axon regeneration in

aged mice was determined. Mice were administered tTA virus and either TRE-OSK virus or

TRE-GFP virus 2 weeks prior to optic nerve crush. Then, fluorescently labeled cholera toxin

B-subunit ß-subunit (CTB) was intraocularly injected into mice that were five weeks instead of two

weeks after optic nerve crush injury. As shown in FIGS. 24A-24B, increasing the length of

time of post-injury OSK expression to five weeks increased the number of estimated axons

per nerve in the 12 month old mice compared to two weeks post-injury of OSK expression in

FIG. 23B. In contrast, increasing the length of time of post injury GFP expression had no

effect on axon regeneration (compare results with GFP in FIGS. 24A-24B with those shown

in FIGS. 23A-23B). Therefore, the data suggests that a longer time of OSK expression may

be beneficial in promoting axon regeneration and RGC survival after nerve crush injury in

aged mice.

Example 10. Induction of OSK expression following optic nerve crush injury increased axon

regeneration and RGC survival in mice.

[00400] It was It was alsoalso determined determined whether whether induction induction of OSK of OSK expression expression after after optic optic nerve nerve

crush injury would promote axon regeneration and RGC survival. Both the Tet-On and Tet-

Off systems depicted in the panel of FIG. 22 were used. In the Tet-On system, AAV virus

with the TRE-OSK-SV40 vector and AAV virus encoding rtTA under the CMV constitutive

promoter were produced through routine methods and injected into the retina of mice. As

depicted in FIG. 25A, in the Tet-On system (top panel), OSK expression was induced by

giving mice doxycycline either prior to optic nerve crush injury or after optic nerve crush

injury. A cohort of mice were not treated with doxycycline as a control (no induction). In

the Tet-Off system, AAV virus with the TRE-OSK-SV40 vector and AAV virus encoding

tTA under the CAG constitutive promoter were produced through routine methods and

injected into the retina of mice. As depicted in FIG. 25A, in the Tet-Off system (bottom

panel), OSK expression was suppressed after optic nerve crush injury. Fluorescently labeled

cholera toxin B-subunit (CTB)injection -subunit (CTB) injectionwas wasused usedto tovisualize visualizeaxons. axons.

[00401] As shown As shown in FIG. in FIG. 25B,25B, induction induction of OSK of OSK expression expression postpost injury injury through through Tet-On Tet-On

system significantly increased the number of estimated axons per nerve compared to no

induction of OSK or induction of OSK prior to injury (pre-injury) only through either Tet-On

or Tet-Off system. Furthermore, induction of OSK expression post injury significantly

increased the survival of RBPMS+ cells compared to no induction of OSK expression or

compared to OSK induction pre-injury only through either Tet-On or Tet-Off system (FIG.

PCT/US2019/053545

25C). Therefore, the Tet-On system depicted in FIG. 25A, top panel, allowed for temporal

control of OSK expression and induction of OSK after optic nerve crush injury promoted

axon regeneration and RGC survival. Without being bound by a particular theory, induction

of OCT4, KLF4, and SOX2 expression using a Tet-Off system following an injury may

promote regeneration when recovery from an injury does not require immediate expression of

OCT4, KLF4, and/or SOX2.

Example 11. Superior effect of OCT4, SOX2, and KLF4 (OSK) expression from a single

transcript compared to individual transcripts in promoting axon regeneration.

[00402] ThisThis example example explored explored the the effect effect of expressing of expressing OCT4, OCT4, SOX2, SOX2, and and KLF4KLF4 under under

one promoter as compared to expression of OCT4, SOX2, KLF4 alone or in combination

under separate promoters. AAV virus encoding tTA under the CAG constitutive promoter

and AAV virus or viruses encoding (1) OCT4 under the TRE promoter, (2) SOX2 under a

TRE promoter, (3) KLF4 under a TRE promoter, (4) OCT4 and SOX2 under one TRE

promoter, (5) OCT4, SOX2, and KLF4 each under separate promoters, or (6) OCT4, SOX2,

and KLF4 under the same promoter were injected into the retina of mice. A schematic

showing the various vectors used in this study is shown in FIG. 26A. Optic nerve crush

injury was induced 2 weeks after virus administration. Fluorescently labeled cholera toxin B- ß-

subunit (CTB) injection 2 weeks after optic nerve crush was used to image axons.

[00403] As shown As shown in FIG. in FIG. 26B,26B, whenwhen all all three three transcription transcription factors factors (OSK) (OSK) werewere expressed expressed

under one promoter, the number of estimated axons per nerve was at least four times higher

than when OCT4, SOX2, and KLF4 were each expressed under a separate promoter (e.g.,

compare OCT4, SOX2, KLF4 (5), and OCT4-SOX2-KLF4 (6) results). Similarly, the

number of estimated axons per nerve was also at least four times higher when OSK was

expressed on a single transcript than when OCT4, SOX2, and KLF4 expression alone (FIG.

26B) (e.g., compare OCT4 (1), SOX2 (2), and KLF4 (3) with OCT4-SOX2-KLF4 (6)

results). The increase in axon regeneration was likely attributed to expression of all three

transcription factors (OSK) under one promoter, as expression of OCT4 and SOX2 under one

promoter did not significantly increase the number of estimated axons per nerve relative to

expression of each transcription factor alone (FIG. 26B) (e.g., compare OCT4-SOX2 (4) with

OCT4-SOX2-KLF4 (6) results).

Analysis

[00404] Analysis of retina of retina ganglion ganglion cellcell (RGC) (RGC) survival survival was was alsoalso conducted conducted by by

quantifying RBPMS+ cells. As shown in FIG. 26C, expression of OSK from one promoter

increased the survival of RBPMS+ cells relative to expression of OCT4, SOX2, or KLF4

WO wo 2020/069373 PCT/US2019/053545

alone and relative to expression of OCT4 and SOX2 under one promoter. Expression of OSK

from one promoter also increased the survival of RBPMS+ cells relative to expression of

OCT4, SOX2, or KLF4 from separate vectors in separate viruses.

As shown

[00405] As shown by the by the fluorescence fluorescence staining staining depicted depicted in FIG. in FIG. 26D,26D, expression expression of of

OCT4, SOX2, and KLF4 in separate vectors in separate viruses resulted in a heterogeneous

population of RGCs. Some cells only expressed OCT4, SOX2, or KLF4. Some cells

expressed a combination of only two out of the three transcription factors and only a few

three-factor positive RGCs were detected (white color cell in the bottom right corner of the

top left panel in FIG. 26D). In contrast, as shown in FIG. 26E, expression of OCT4, SOX2,

and KLF4 from a single vector resulted in a more homogenous population. All of the cells

expressed all three of the OSK transcription factors (white color cells in the top left panel).

Even in cells that were not pure white, expression of all three transcription factors were

detected as shown in FIG. 26E, suggesting that the results were due to differences in staining

intensity for the three transcription factors.

[00406] Therefore, Therefore, thisthis example example shows shows thatthat expression expression of OCT4, of OCT4, SOX2, SOX2, and and KLF4KLF4 using using

one promoter had greater therapeutic effect (e.g., increased axon regeneration and a greater

survival of retina ganglion cells) compared to expression of each transcription factor alone,

expression of all three transcription factors under separate promoters, or expression of only

two of the transcription factors (e.g., OCT4 and SOX2) under one promoter.

Example 12. Knockdown of Tetl or Tet2 abrogated OSK-induced axon regeneration

following optic nerve crush injury.

[00407] ThisThis example example determined determined the the effect effect of knocking of knocking downdown DNA DNA demethylases demethylases Tet1Tet1

and Tet2 on OSK-induced axon regeneration. A Tet-Off system was used. AAV2 of CAG-

tTA+TRE-OSK-SV40 were injected into mice through intravitreal injection two weeks

before crush together with AAV2 of U6-shRNA. Mice were one month old with four mice in

each group.

[00408] Addgene

[00408] Addgene AAVAAV plasmids plasmids encoding encoding shRNA shRNA sequences sequences were were used. used. Control Control shRNA shRNA

comprised the sequence 5'-GTTCAGATGTGCGGCGAGT-3' (plasmid #85741 from

Addgene). mTET1 (Tet1 shRNA) comprised the sequence 5'-

GCTCATGGAGACTAGGTTTGG-3' (plasmid #85742 from Addgene). mTet2 (Tet2

shRNA) comprised the sequence 5'-GGATGTAAGTTTGCCAGAAGC-3 5'-GGATGTAAGTTTGCCAGAAGC-3'(Plasmid (Plasmid#85743 #85743 from Addgene).

[00409] As shown in FIG. 27, knockdown of either Tet1 or Tet2 significantly reduced the

number of estimated axons per nerve in animals also treated with OSK virus and subjected to

optic nerve crush injury compared to the control hairpin (sh-cntl).

[00410] These These results results suggest suggest thatthat Tet Tet DNA DNA methylases methylases may may be involved be involved in OSK-induced in OSK-induced

axon regeneration and overexpression of Tet (e.g., Tet1 or Tet2) alone or in combination with

OSK expression may promote regeneration.

[00411] As a non-limiting example, mTet3 comprising the sequence 5'-

GCTCCAACGAGAAGCTATTTG-37 GCTCCAACGAGAAGCTATTTG-3' (Plasmid #85740 from Addgene) may be used to knockdown Tet3.

Example 13. Expression of OSK reversed age-related decline in visual acuity and reversed

age-related decline in retina ganglion cell (RGC) function.

[00412] To determine whether age-related visual acuity loss may be reversed with OSK

expression, an optomotor response (OMR) assay was conducted on adult mice (3 month old

mice) and aged mice (12 month old and 18 month old mice). OMR is a reflexive head

movement used to assess visual acuity. To induce OMR, individual mice are placed on a

platform in the middle of an arena surrounded by computer monitors displaying stripes. The

rotation of the striped pattern elicits mouse head tracking in the same direction by reflexive

neck movements. Tracking is monitored by two independent masked observers. Visual

acuity is quantified by increasing the spatial frequency of the stripes until an OMR cannot be

elicited. elicited.

[00413] MiceMice werewere retinally retinally injected injected withwith AAV AAV virus virus encoding encoding tTA tTA and and AAV AAV virus virus

encoding TRE-OSK in the absence of doxycycline (OSK induction condition). In this Tet-

Off system, OSK is expressed from a single promoter in the absence of doxycycline. As

controls, age-matched mice were administered virus encoding virus encoding rtTA and virus

TRE-OSK in the absence of doxycycline (uninduced control, ctl). In the control Tet-On

system, OSK expression requires doxycycline treatment. Adult mice (3 month old (3m))

were also used as a control. An OMR study was conducted to measure the spatial frequency

threshold one month after virus injection.

[00414] As shown As shown in FIG. in FIG. 28, 28, in the in the absence absence of OSK of OSK expression expression (control (control (ctl) (ctl) condition) condition)

the aged mice (12 month old and 18 month old mice) had vision loss compared to the adult

mice (3 month old mice). The decrease in the spatial frequency threshold for the aged mice

relative to the 3 month old mice indicated vision loss in the absence of OSK expression.

When OSK was expressed, however, the spatial frequency threshold on average increased for

WO wo 2020/069373 PCT/US2019/053545

the 12 month old and 18 month old mice relative to no OSK expression. Furthermore, the

spatial frequency thresholds for the 12 month old and 18 month old mice with OSK

expression were similar to that of the 3 month old control mice in the presence and absence

of OSK expression. These results demonstrate that induction of OSK expression reversed

age-related vision loss in mice.

[00415] To determine whether age-related decline in retina ganglion cell (RGC) function

could also be reversed by OSK treatment, electrical waves from RGCs were measured using

pattern electroretinograms (pattern ERGs or pERGs). In pERG assays, a checkerboard light

and dark pattern stimulus is projected via electrodes placed on the cornea of mice of various

ages (3 month old, 12 month old, or 18 month old mice). A contrast reversing pattern is

displayed with no overall change in luminance. Electrical waves generated from the RGCs

are then measured.

[00416] Mice were retinally injected with AAV virus encoding tTA and AAV virus

were also used as a control. A pERG study was conducted to measure the amplitude of the

electrical waves in the RGCs following the pattern stimulus one month after virus injection.

As shown

[00417] As shown in FIG. in FIG. 29, 29, electrical electrical waves waves generated generated fromfrom RGCsRGCs declined declined in aged in aged

mice (3 month old mice compared to 12 month old and 18 month old mice) in the absence of

OSK expression (ctl condition). In contrast, administration of AAV virus encoding tTA and

AAV virus encoding TRE-OSK in the absence of doxycycline (OSK induction condition)

restored RGC electrical waves in 12 month old mice. For 18 month old mice, however, RGC

function was likely not restored because corneal opacity blocked the pattern stimulus. These

results suggest that expression of OSK improved RGC function in aged (12 month old) mice.

Therefore,

[00418] Therefore, thisthis example example demonstrates demonstrates thatthat induction induction of OSK of OSK expression expression can can

improve vision acuity and RGC function that is caused by aging.

Example 14. Expression of OSK reversed glaucoma-induced decline in visual acuity and

reversed glaucoma-induced decline in retina ganglion cell (RGC) function.

[00419] To determine To determine whether whether OSK OSK expression expression could could be used be used to reverse to reverse glaucoma- glaucoma-

induced declines in visual acuity and RGC function, a mouse model of glaucoma was used.

Chronic elevation of intraocular pressure (IOP) was induced unilaterally in adult C57BL/6J

mice by injecting polystyrene microbeads to the anterior chamber. IOP was measured in the

first four weeks. As shown in FIG. 30A, microbead injection increased IOP 4-21 days after

microbead injection. Axon density was quantified using p-phenylenediamine (PPD) staining

(FIG. 30B). FIG. 30C includes a chart quantifying RGC cell density (left panel) using Brn3a

staining (shown, for example, on the right). FIGS. 30B-30C show that 4 weeks after

microbeads injection into the anterior chamber of the eye, there was significant loss of axon

density and RGC density in wild-type (WT) mice that were not treated with AAV virus

encoding TRE-OSK.

[00420] In these In these experiments, experiments, glaucoma glaucoma was was induced induced withwith microbead microbead injection injection and and thenthen

three weeks later, OMR and pERG assays were conducted (pre AAV injection measurements

in FIGS. 30D-30E). Then, mice were divided into two treatment groups. One group of mice

were retinally injected with AAV virus encoding rtTA and AAV virus encoding TRE-OSK in

the absence of tetracycline (OSK AAV OFF) or with AAV virus encoding tTA and AAV

virus encoding TRE-OSK (OSK AAV ON). Four weeks post AAV virus injection, OMR and

pERG assays were conducted again (4w post AAV) measurements in FIGS. 30D-30E). As a

control, experiments were also conducted with injection of saline instead of microbeads (no

glaucoma control).

[00421]

[00421] As As shownininFIG. shown FIG. 30D, 30D, induction inductionofofOSK expression OSK (OSK(OSK expression AAV ON) AAV increased ON) increased

the spatial frequency threshold compared to no induction of OSK expression (OSK AAV

OFF) for mice with glaucoma (mice injected with microbeads). These results suggest that

induction of OSK expression can improve glaucoma-related vision loss.

[00422] As shown As shown in FIG. in FIG. 30E,30E, induction induction of OSK of OSK expression expression restored restored the the electrical electrical wavewave

amplitude in mice with microbead-induced glaucoma. These results suggest that induction of

OSK expression can also reverse glaucoma-related decline in RGC function.

Therefore,

[00423] Therefore, induction induction of OSK of OSK expression expression can can improve improve the the symptoms symptoms induced induced by by

glaucoma.

Example 15. Expression of human OSK promoted survival of human neurons and axon

regrowth following vincristine-induced neuronal damage.

[00424]

[00424] To To determine determine whether whether expression expression of of human human OCT4, OCT4, human human KLF4, KLF4, andand human human

SOX2 (human OSK) could protect human neuronal cells and regenerate axons in vitro, a

neurite regeneration assay was used as described below. SH-SY5Y cells, which are human

neuroblastoma cells, were differentiated into neurons and were transduced with a AAV.DJ

WO wo 2020/069373 PCT/US2019/053545

vector encoding human OCT4, human KLF4, and human SOX2 under a Tet-inducible

promoter (using a Tet-Off system). In the OSK Off condition, OSK expression was not

induced in cells. In the OSK On condition, OSK expression was induced in cells. Five days

after transduction, vincristine (VCS) was used to induce neurite degeneration. Cells were

treated with VCS for 24 hours or 48 hours. A schematic of a treatment timeline (with 24 hour

VCS treatment) is provided in the left portion of FIG. 31A. VCS is a chemotherapy drug that

disrupts microtubules. It is often used in vitro to determine whether treatments maintain

and/or promote cellular function (e.g., neuronal function) after damage. As described herein,

VCS was used determine the effect of OSK treatment on neuronal survival and axon

regrowth. After VCS treatment, cells were grown in differentiation medium and neurite

outgrowth was assayed.

[00425] In FIG. 31A,31A, In FIG. cells werewere cells assayed for for assayed neuronal outgrowth neuronal ninenine outgrowth daysdays after cells after werewere cells

treated with VCS for 24 hours. Cells in which OSK expression was induced (OSK On

condition) showed increased neuronal survival and axon outgrowth relative to cells in which

OSK expression was not induced (OSK Off condition) (FIG. 31A). Quantification of

neuronal cell area similarly showed that OSK expression increased the cell area of neurons by

at least 8 times compared to no OSK expression (FIG. 31B). Similar results were also

observed with 48 hours of VCS treatment (FIG. 31C).

[00426] These results show that expression of human OSK protected human neuron cells

against VCS-induced neuron degeneration.

Methods Cell Culture and Differentiation Protocol

[00427] SH-SY5Y neuroblastoma cells were obtained from the American Tissue Culture

Collection (ATCC, Collection (ATCC,CRL-2266) and and CRL-2266) maintained according maintained to ATCCtorecommendations. according The ATCC recommendations. The

cells were cultured in a 1:1 mixture of Eagle's Minimum Essential Medium (EMEM, ATCC,

30-2003) and F12 medium (ThermoFisher Scientific, 11765054), supplemented with 10%

fetal bovine serum (FBS, Sigma, F0926) and 1 X penicillin/streptomycin (ThermoFisher

Scientific, 15140122). Cells were cultured at 37 °C with 5% CO2 and 3% CO and 3% O. O2. Cells Cells were were

passaged at ~80% confluency.

[00428] SH-SY5Y cells were differentiated into neurons as previously described (Encinas

et al., J Neurochem. 2000 Sep;75(3):991-1003; Sep:75(3):991-1003; Shipley et al., J Vis Exp. 2016 Feb

17;(108):53193), with some modifications. Briefly, 1 day after plating, cells started to be

WO wo 2020/069373 PCT/US2019/053545

differentiated in EMEM/F12 medium (1:1) containing 2.5% FBS, 1x penicillin/streptomycin,

and 10 M µMall-trans all-transretinoic retinoicacid acid(ATRA, (ATRA,Stemcell StemcellTechnologies, Technologies,72264) 72264)(Differentiation (Differentiation

Medium 1) for 3 days, followed by treating the cells in EMEM/F12 (1:1) containing 1% FBS,

1 X penicillin/streptomycin, and 10 M µMATRA ATRA(Differentiation (DifferentiationMedium Medium2) 2)for for33days. days.Cells Cells

were then split into 35mm cell culture plates coated with poly-D-lysine (ThermoFisher

Scientific, A3890401). One day after splitting, neurons were matured in serum-free

neurobasal/B27 plus culture medium (ThermoFisher Scientific, A3653401) containing 1 X

Glutamax (ThermoFisher Scientific, 35050061), 1 X penicillin/streptomycin, and 50 ng/ml

BDNF (Alomone labs) (Differentiation Medium 3) for at least 5 days.

Neurite regeneration assay

[00429] TheThe

[00429] differentiated neurons differentiated neurons from fromSH-SY5Y SH-SY5Ycells were cells transduced were with AAV.DJ transduced with AAV.DJ 6 vectors at 10 genome copy per cell. Five days after transduction, 100 nM vincristine (Sigma,

V8879) was added to the cells for 24 hours or 48 hours to induce neurite degeneration. After

vincristine treatment, neurons were washed in PBS twice and fresh differentiation medium

was added back to the plates. Neurons were followed for neurite outgrowth for up to 2 weeks.

Example 16. Recovery from injury and restoration of vision by Tet-dependent resetting of the

epigenetic clock.

To determine

[00430] To determine whether whether mammalian mammalian cells cells might might retain retain a faithful a faithful copycopy of epigenetic of epigenetic

information from earlier in life, it was tested whether the three gene combination of OSK was

sufficient to reset age. The three-gene OSK combination into fibroblasts from old mice and

measured its effect on RNA levels of genes known to be altered with age, such as H2A, H2B,

LaminB1. and LaminB and Chaf1b. Chaflb. OSK OSK treatment treatment ofof fibroblasts fibroblasts from from old old mice mice restored restored youthful youthful gene gene

expression patterns, expression patterns, similar similar to what to what OSKM does, OSKM does, with nowith no apparent apparent loss ofidentity loss of cellular cellularor identity or

the induction of Nanog, an early embryonic transcription factor that can induce teratomas

(FIG.36A-36C).

To deliver

[00431] To deliver and and control control OSK OSK expression expression in vivo, in vivo, a tightly a tightly regulated regulated Tet-ON Tet-ON and and

Tet-OFF adeno-associated viral (AAV) vector system was developed to accommodate all

three reprogramming genes in one viral particle (Smalley et al., First AAV gene therapy

poised for landmark approval. Nat Biotechnol, 2017. 35(11): p. 998-999; Senis et al., AAV

vector-mediated in vivo reprogramming into pluripotency. Nat Commun, 2018. 9(1): p. 2651)

(FIG. 32A). First, to test if induction of OSK AAVs caused toxicity in vivo, 5-month-old

C57BL/6J mice were infected with rtTA and TRE-OSK AAV9s and induced expression to levels comparable to those of transgenic mice (FIG. 36D). Surprisingly, continuous induction of OSK for over a year had no discernable negative effect on the mice for over a year (FIG.

32B and FIG. 36E). Without being bound by a particular theory, there was ostensibly no

discernable negative effect on the mice because high-level expression in the intestine was

avoided (FIGs. 36F-36H), thus avoiding the dysplasia and weight loss seen in other studies,

including Abad et al., Nature 502, 340-345, doi:10.1038/nature12586 doi: 10.1038/nature12586(2013). (2013).

Almost

[00432] Almost allall speciesexperience species experience a decline declineininregenerative potential regenerative during potential ageing. during In ageing. In

mammals, one of the first systems to lose this potential is the central nervous system (CNS).

A canonical CNS cell type, the retinal ganglion cell, projects an axon away from the retina

towards the brain, forming the optic nerve. During embryogenesis and in neonates, RGCs can

regenerate if damaged, but this capacity is soon lost (Goldberg et al., Science, 2002.

296(5574): p. 1860-4). Over time, as organisms age, the overall function and resilience of the

CNS continues to decline (Geoffroy et al., Cell Rep, 2016. 15(2): p. 238-46). To explore

whether it is possible to restore an early epigenetic profile in adult RGCs, OSK expression

was induced in a nerve crush injury model in adult mice of various ages. The Tet-Off system

(Tet-Offtta-AAV2) (Tet-Off tTA-AAV2)carrying carryingOSK, OSK,either eitherin inseparate separateAAVs AAVsor orin inthe thesame sameAAV, AAV,was was

injected into the vitreous body, resulting in efficient, selective, and doxycycline-responsive

gene expression in RGCs. As a negative control, a group of mice were also treated with

doxycycline to repress the AAVs (FIG. 32C and FIG. 37C). Two weeks post-injection, optic

nerve crush was performed, and, two weeks after that, axon length and optic nerve density

were calculated (FIG.32D).

Induction

[00433] Induction of the of the polycistronic polycistronic OSK-AAV2 OSK-AAV2 caused caused a significant a significant increase increase in RGC in RGC

survival and long-distance axonal regeneration (FIG. 32E and FIG. 37D) without any sign of

RGC proliferation (FIG. 38A). In contrast, when introduced on separate AAVs, OCT4,

SOX2, KLF4 had no effect on regenerative capacity (FIG. 32E), ostensibly due to the lower

frequency of co-infection (FIG. 37A and FIG. 37B). Because Klf4 can repress axonal growth

(Moore et al., Science, 2009. 326(5950): p. 298-301; Qin et al., Nat Commun, 2013. 4: p.

2633), OCT4, SOX2, and KLF4 were also individually and a dual-cistron of Oct4 and Sox2

was tested. No regenerative effect, however, was observed in the absence of Klf4.

Remarkably, if poly-cistronic OSK was induced for 3-months, RGC axon fibers extended all

the way to the chiasm, a distance of over 3 mm (FIG. 38B). Indeed, when polycistronic OSK

was induced for 12-16 weeks, regenerating RGC axon fibers further extended into the chiasm

(5 mm away from crush site), where optic nerve connects to brain (FIGs. 38B-38C).

WO wo 2020/069373 PCT/US2019/053545

[00434] Next, the requisite timing of OSK expression was tested to promote neuronal

survival and regeneration. For these experiments, the Tet-On AAV system was utilized due

to its rapid on-rate (FIG. 37D and FIGS. 39A-39B). Significant improvement in axon

regeneration only occurred when OSK expression was induced after injury and the longer

OSK was induced, the greater distance the neurons extended, with no increase in the total

number of RGCs (FIGS. 33B, 33C, and 33D). By co-staining for OSK and performing

neuronal counts, survival rate was estimated to be 2.5-3 times of uninfected or GFP-infected

RGCs (52 vs. 17%-20%) (FIGS. 39C and 39D), suggesting OSK effect is cell-intrinsic. The

Pten-mTOR-S6K pathway, previously shown to improve neuronal survival in vivo, was not

activated in OSK-infected cells post-injury (FIG. 40A and FIG. 40B), indicating a new

pathway might be involved.

[00435] It was It was determined determined whether whether neuronal neuronal injury injury advanced advanced epigenomic epigenomic age age and and whether whether

OSK's benefits were due to the preservation of a younger epigenome. Genomic DNA from

RGCs was FACS-isolated before injury or 4-days after injury in the presence or absence of

OSK induction, and subjected reduced-representation bisulfite sequencing (RRBS-Seq).

Without being bound by a particular theory, rDNAme clock (Wang et al., Genome Res 29,

325-333, oi:10.1101/gr.241745.118 (2019)) doi: 10.1101/gr.241745.118 provided (2019)) the provided best the site best coverage site (70/72 coverage CpG (70/72 CpG

sites) relative to other available mouse clocks (Meer et al., Elife 7, doi: :10.7554/eLife.40675 10.7554/eLife.40675

(2018); Thompson et al., Aging (Albany NY) 10, 2832-2854, bi:10.18632/aging 101590 doi: 10.18632/aging.101:

(2018)) and its age estimate remained highly correlated with chronological age of RGCs

(FIG. 45A and Methods). In the absence of global methylation changes, injured RGCs

experienced an acceleration of the epigenetic clockand OSK expression counteracted this

effect (FIG. 33K and FIG. 45B).

[00436] It was It was determined determined whether whether thatthat the the effect effect of OSK of OSK on neuronal on neuronal survival survival and and

regeneration occurred by restoring a younger epigenome. If so, these effects should be

dependent on the reversal of the epigenetic clock, which would require the removal of methyl

groups from DNA via the activity of Ten-Eleven-Translocation (TET) dioxygenases.

Previously characterized AAVs expressing short-hairpin RNAs against Tet1 and Tet2 (sh-

Tet1 and sh-Tet2) (Guo et al., Cell 145, 423-434, doi: 10.1016/j.cell.2011.03.022 (2011); Yu

et al., Nat Neurosci 18, 836-843, bi:10.1038/nn.4008 (2015); doi: 10.1038/nn.4008 Weng (2015); et et Weng al., Neuron al., 94, Neuron 337- 94, 337-

346.e336, doi: 10.1016/j.neuron.2017.03.034 (2017)) were utilized, and the transduction rate

and knockdown efficiency in vivo was validated (FIGs. 40C-40F). Knockdown of either Tet1

or Tet2 (sh-Tet1 and sh-Tet2 AAV2, at 1/5 titer of OSK AAV), which transduced around

WO wo 2020/069373 PCT/US2019/053545

70% of OSK positive cells (FIGs. 40C and 40D), efficiently blocked OSK from regenerating

axons and improved RGC survival (FIGS. 33E and 33F).

To test

[00437] To test whether whether neuronal neuronal rejuvenation rejuvenation by OSK by OSK is specific is specific for for mouse mouse RGCs, RGCs, axonaxon

regeneration assays were performed in human neurons in vitro (FIG. 33G). Human

neuroblastoma SH-SY5Y cells were differentiated into neurons and transduced them with

AAV-DJ vectors to express OSK (FIG. 33G, FIG. 41A, and FIG. 41B). Similar to mouse

RGCs in vivo (FIG. 38A), OSK did not induce cell proliferation (FIGs. 41C-41D). Axon

degeneration was then induced by a 24 hour treatment with vincristine (VCS), a

chemotherapeutic agent, and cells were then allowed to recover for 9 days. The epigenetic

clock of these neurons were measured using the skin and blood cell clock (Horvath and Raj,

Nat Rev Genet. 2018 Jun; 19(6):371-384). Similarly, DNA methylation age is significantly

increased after VCS damage in human neurons (FIG. 41J), and OSK expression not only

prevented this increase of DNA methylation age, but also restored a younger DNA

methylation age without a global reduction of DNA methylation (FIG. 33H, bottom panel and

FIG. 45C). DNAmAge is significantly decreased with experiment day 9 post VCS damage in

OSK treated cells, but not in cells not treated by OSK (FIG.33H). At Day 9 post damage, the

neurite area was 15-fold greater in the rejuvenated OSK-transduced cells than controls (FIG.

41E and FIG. 41F) and the recovery from damage was dependent on the Tet2 demethylase

(FIG.33I, (FIG.331, FIG. 33J, and FIG. 41G), even in presence of high OSK expression (FIG. 41K) but

not the mTOR-S6K pathway, paralleling mouse retinal ganglia cells (FIG. 41H and FIG.

41I). 411). Thus, the ability of OSK to reprogram neurons and promote axon growth is cell

intrinsic, conserved in mammals, and requires epigenetic rejuvenation through DNA

demethylation. This process is referred to herein as the recovery of information via

epigenetic reprogramming, or "REVIVER" for short.

Glaucoma,

[00438] Glaucoma, a progressive a progressive lossloss of RGCs of RGCs and and their their axons, axons, mostmost often often due due to to

increased intraocular pressure, is a leading cause of age-related blindness worldwide.

Although some treatments can slow down disease progression, it is currently not possible to

restore vision once it has been lost. Given the ability of OSK to regenerate axons after acute

nerve damage, we decided to test whether REVIVER treatment could restore the function of

RGCs in achronic setting like glaucoma (FIG. 34A). Elevated intraocular pressure (IOP)

was induced unilaterally for 4-21 days by injection of microbeads into the anterior chamber.

OSK AAVs or PBS were then injected intravitreally, and express at a time point when

glaucomatous damage was established, with a significant decrease in RGCs and axonal

density (FIG. 34B, FIG. 42A, and FIG. 42B) ( Krishnan et al., J Immunol, 2016. 197(12): p.

WO wo 2020/069373 PCT/US2019/053545

4626-4638). At four weeks post-AAV injection, OSK-On treated mice presented with a

significant increase in axon density when compared to PBS and OSK-Off treated mice. The

increased axon density observed was equivalent to the axon density in the saline-only, non-

glaucomatous mice (FIGS. 34C and 34D), and was not associated with proliferation of RGCs

(FIG. 42C).

[00439] To determine To determine whether whether the the increased increased axonaxon density density observed observed in OSK in OSK treated treated micemice

coincided with increased vision, a behavior assay, optomotor response (OMR), was used

(FIG 34E) to track the visual acuity of each mouse. Compared to mice that received either

PBS or the OSK-Off AAV, OSK treatment significantly increased visual acuity relative to the

pre-treatment baseline measurement, restoring more than half of the vision loss (FIG. 34F). A

readout of electrical waves generated by RGCs in response to a reversing contrast

checkerboard pattern, known as Pattern electroretinogram response (pERG) analysis, showed

that OSK treatment significantly improved RGC function relative to the pre-treatment

baseline measurements, as well as, compared with either PBS or OSK-Off AAV treated mice

(FIG. 34G and H). Without being bound by a particular theory, treatment with OSK AAV, as

shown herein, may be the first treatment to reverse vision loss in any glaucoma model.

Notably, OSK reversed vision loss in a glaucoma model.

[00440] Given the ability of OSK to induce axon regeneration following optic nerve crush

and to restore vision after glaucomatous damage in young mice, it was determined whether

OSK could also restore vision loss associated with physiological aging and regenerate axons

following optic nerve injury in aged mice. This is particularly important since a recently

reported retinal rod photoreceptor regenerative approach that was successful when treating

young mice was significantly diminished when treating older mice (Yao, K., et al.,

Restoration of vision after de novo genesis of rod photoreceptors in mammalian retinas.

Nature, 2018. 560(7719): p. 484-488.).

[00441] To To

[00441] determinewhether determine whether OSK OSK AAV AAV treatment treatmentcould induce could axonaxon induce regeneration in regeneration in

aging mice, the optic nerve crush injury model was performed on 12-month-old mice using

the same protocol as in FIG. 32D with the experimental design shown (FIG. 35A). In aged

mice, OSK AAV treatment for two weeks post-injury showed doubled RGC survival, similar

to that observed in young mice (FIG. 43A). Though the axon regeneration is slightly less than

young mice at two weeks post injury (FIG. 43B), OSK AAV treatment in aged mice for five

weeks post-injury showed a significant increase in axon regeneration (FIGS. 35B and 35C),

similar to that observed in young mice. These data indicate that aging does not diminish the

PCT/US2019/053545

effectiveness of OSK AAV treatment in inducing axon regeneration following an optic nerve

crush injury.

[00442] To test whether OSK treatment could reverse vision loss associated with

physiological aging, 4- and 12-month-old mice received intravitreal injections of OSK-Off or

OSK-On AAV. As expected, at one year of age, mice showed a significant reduction in visual

acuity and RGC function as measured by OMR and pERG, which was restored by OSK AAV

treatment (FIG.35D and FIG. 43C). Such restoration was not observed in 18 month-old mice

(FIG. 43F-43G) likely due to spontaneous corneal opacity developed at this age (McClellan

et al., Am J Pathol 184, 631-643, doi: 10.1016/j.ajpath.2013.11.019 (2014)), suggesting the

restoration effect is specifically contributed by AAV-infected RGC layer.

[00443] Next, Next, it was it was determined determined whether whether restoration restoration of youthful of youthful transcriptome transcriptome by OSK by OSK

indicates a youthful epigenome and thus would requires Tet enzymes. Remarkably, Tet1 or

Tet2 knockdown completely blocked the rejuvenation effect of OSK-On AAV treatment as

measured by both OMR and pERG analyses (FIG.35E and FIG. 35F), consistent with DNA

methylation as the key process for vision restoration. Notably, there is no obvious RGC and

axon density increase by OSK in aged mice (FIG. 43D and FIG. 43E), suggesting functional

improvement of existing RGCs. The rDNA methylation age of FACS-sorted RGCs from 12

month-old mice was measured. OSK AAV expression for 4 weeks significantly decreased the

DNA methylation age and Tet1 and Tet2 knockdown blocked such rejuvenation (FIG. 35I). 351).

Together, these results demonstrate that Tet-dependent in vivo reprogramming can restore

youthful gene expression patterns, reverse the DNA methylation clock, and restore the

function and regenerative capacity of a complex tissue.

[00444] To further determine whether Tet2 knockout can block the effect of OSK on axon

regeneration, mouse OSK and Tet2 conditional knockout mice (B6;129S-Tet2tm1.1Iaai/J)

were used. Mouse eyes were injected with (1) AAV-CRE (Tet2 cKO); (2) AAV-tTA +

AAV-TRE-OSK: OSK (Tet2 WT); or (3) AAV-tTA + AAV-TRE-OSK + AAV-CRE: OSK (Tet2cKO) After (Tet2 cKO). two After weeks, two optic weeks, nerve optic crush nerve was crush conducted. was CTB conducted. was CTB administered was two administered two

weeks after optic nerve crush and mice were sacrificed two days after CTB administration to

determine the extent of axon regeneration following injury. As shown in FIGs. 46A-46B, the

number of axons per nerve up to at least 500 um µm from the injury site was significantly higher

in Tet2 wild-type mice that were administered OSK as compared to Tet2 knockout mice that

were administered OSK. These results suggest that OSK-mediated axon regeneration is

Tet2-dependent.

WO wo 2020/069373 PCT/US2019/053545

[00445] In order to determine the effect of reprogramming on the transcriptome in the

retina, FACS-purified RGCs from intact old mice (12 month) and those that were either

treated with empty control AAV (TRE-OSK) or OSK-On (tA+TRE-OSK) were analyzed by

genome-wide RNA-seq. Compared to RGCs from intact young mice (5 month), 464 genes

were identified that were differentially-expressed during ageing (FIG. 35G, FIG. 35I, 351, FIG.

44A, and Table 5) and not induced by empty AAV alone. Of these, 268 genes were down-

regulated during aging which were enriched in sensory perception genes (FIG.35I), (FIG.351),

suggesting a decline of signaling receptors/sensory function during aging (FIGS. 44B and

44C). Interestingly, 116 of these genes appear uncharacterized, lacking an official gene name.

The other 196 genes that are slightly up-regulated during aging are enriched of ion transporter

genes (FIG. 44D).

[00446] Remarkably, Remarkably, consistent consistent withwith OSK OSK resetting resetting the the epigenomic epigenomic landscape, landscape, the the vastvast

majority (90%, 418) of the 464 genes that change in expression during aging were restored

towards youthful levels after treatment (FIGS. 35G and 35H). Together, these results

demonstrate that Tet-dependent in vivo reprogramming can restore youthful gene expression

patterns, reverse the epigenetic clock, and restore the function of a tissue as complex as the

retina.

[00447] Post-mitotic neurons in the central nervous system are some of the first cells in

the body to lose their ability or regenerate. In this study, it was shown that in vivo

reprogramming of aged neurons can reverse epigenetic age and allow them to regenerate and

function as though they were young again. The requirement of the DNA demethylases Tet1

and Tet2 for this process indicates that DNA methylation at clock sites are not merely an

indicator of ageing, but an active participant in it. It was concluded that mammalian cells

retain a set of original epigenetic information, in the same way Shannon's observer stores

information to ensure the recovery of lost information at a later time (SHANNON, C.E., A

Mathematical Theory of Communication. The Bell System Technical Journal, 1948. 27: p.

379-423). How cells are able to find and remove the appropriate DNA methylation moieties

and restore youthful gene expression patterns is still an open question, but even in the

absence of this knowledge, our data indicate that the reversal of epigenetic age could be an

effective translational strategy, not just to restore vision, but to give other tissues the ability to

recover from injury and resist age-related decline.

wo 2020/069373 WO PCT/US2019/053545

Table 5. Genes that were differentially expressed during ageing in mice RGCs.

Downregulated genes Upregulated Genes 1700031P21Rik 0610040J01Rik 1810053B23Rik 1700080N15Rik 2900045O20Rik 2900064F13Rik 2900064F13Rik 2900060B14Rik 4833417C18Rik 4921504E06Rik 4921522P10Rik 4921522P10Rik 4930402F11Rik 4930447C04Rik 4930453C13Rik 4930488N15Rik 4930455B14Rik Ace 4930500H12Rik Ackr1 4930549P19Rik 4930549P19Rik Acot10 4930555B11Rik Acvrl 4930556J02Rik Adamts17 4932442E05Rik 4932442E05Rik Adralb 4933431K23Rik AI504432 4933438K21Rik Best3 Best3 6720475M21Rik Boc 9830132P13Rik Cadm3 A430010J10Rik Cand2 A530064D06Rik Ccl9 A530065N20Rik Cd14 Abcb5 Cd36 Abhd17c Cfh Cfh AC116759.2 Chrm3 AC131705.1 Chrna4 AC166779.3 Cntn4 Acot12 Cracr2b Adig Cryaa Akr1cl Akr1cl CT573017.2 Ankrd1 Cyp26a1 Cyp26a1 Asb15 Cyp27a1 Cyp27a1 Atp2c2 D330050G23Rik AU018091 D930007P13Rik AW822073 Ddo Btnl10 Btnl10 Dgkg C130093G08Rik Dlk2 C730027H18Rik Dnajal-ps Dnajal-ps Ccdc162 Ccdc162 Drd2 Chil6 Dsel Dsel Col26a1 Dytn Corin Corin Ecscr Crls1 Crls1 Edn1

166

Cybrd1 Ednrb Cyp2d12 Efemp1 Cyp7a1 Cyp7a1 Elfn2

D830005E20Rik Ephal0 Epha10 Dlx3 Ephx1 Dnah14 Erbb4 Dsc3 Dsc3 Fam20a Dthd1 Fbxw21 Eid2 Ffar4 Eps811 Flt4

EU599041 Fmod Fam90a1a Foxp4 Fancf Fzd7 Fau-ps2 Gabrd Fezf1 Galnt15 Gja5 Galnt18 Gm10248 Gfra2 Gm10513 Ggtl Ggt1 Gm10635 Gm10416 Gm10638 Gm14964 Gm10718 Gm17634 Gm10722 Gm2065 Gm10800 Gm32352 Gm10801 Gm33172 Gm11228 Gm34280 Gm11251 Gm35853 Gm11264 Gm36298 Gm11337 Gm36356 Gm11368 Gm36937 Gm11485 Gm3898 Gm11693 Gm42303 Gm12793 Gm42484 Gm13050 Gm42537 Gm13066 Gm42743 Gm13323 Gm43151 Gm13339 Gm43843 Gm13346 Gm44545 Gm13857 Gm44722 Gm14387 Gm45516 Gm14770 Gm45532 Gm15638 Gm47494 Gm16072 Gm47982 Gm16161 Gm47989

Gm16181 Gm48398 Gm17200 Gm48495 Gm17791 Gm48593 Gm18025 Gm48958 Gm18757 Gm49089 Gm18795 Gm49326 Gm18848 Gm49331 Gm49331 Gm19719 Gm49760 Gm20121 Gm5796 Gm20356 Gm6374 Gm2093 Gm7276 Gm21738 Gm8237 Gm21940 Gm9796 Gm22933 Gm9954 Gm24000 Gpr75

Gm24119 Gprc5c

Gm25394 Grid2ip

Gm26555 Gsg112

Gm27047 Hapln4

Gm28262 Hcn3 Gm28530 Hcn4 Gm29295 Hhatl

Gm29825 Hs6st2

Gm29844 Htr3a Illrap Gm3081 Il1rapl2 Gm32051 Gm32122 Inkal

Gm33056 Kbtbd12

Gm33680 Kcnj11

Gm34354 Kcnk4 Gm34643 Kdelc2

Gm3551 Klhl33 Klhl33 Gm36660 Lamc3 Lilra5 Lilra5 Gm36948 Gm37052 Lman11 Gm37142 Lrfn2

Gm37262 Lrrc38

Gm37535 Lrrn4cl

Gm37569 Ltc4s

Gm37589 Mansc1 Gm37647 Mir344c

Gm37648 Msr1 Msr1 Gm37762 Mycbpap

Gm38058 Myoc Gm38069 Ngfr

Gm38137 Nipal2 Nipal2 Gm38218 Olfr1372-ps1

Gm39139 Otop3

Gm42535 P2rx5

Gm42680 P2ry12

Gm42895 P4ha2

Gm42994 Pcdha12

Gm43027 Pcdha2

Gm43158 Pcdhac2

Gm43288 Pcdhb18

Gm43366 Pcdhb5

Gm44044 Pcsk2os1

Gm44081 Pcsk6

Gm44187 Perp

Gm44280 Pkp1

Gm44535 Plxna4

Gm45338 Prickle2

Gm45644 Qsox1

Gm45740 Rapgef4os2

Gm46555 Rbp4 Gm46565 Rcn3 Gm4742 Sec1415

Gm47485 Sel113

Gm47853 Serpinh1

Gm47992 Sgpp2 Sgpp2 Gm48225 Shisa6

Gm48314 Siah3

Gm48383 Siglech

Gm48673 Slc12a4

Gm48804 Slc24a2

Gm48832 Slc2a5

Gm4994 Slc4a4 Slitrk3 Gm5487 Gm5724 Smagp Smagp Gm595 Smoc2 Smoc2 Gm6012 Speer4b

Gm6024 Spon2 Gm7669 Sstr2 Sstr3 Gm7730 Gm8043 St3gal3

Gm8953 Stcl Stc1

Gm9348 Stc2

Gm9369 Syndig1 Gm9495 Syt10 H2al2a Thsd7a Ido2 Tlr8

Igfbp1 Tmem132a Kif7 Tmem132d Klhl31 Tmem200a Lrrc31 Tmem44 Mc5r Trpc4

Mgam Trpv4 Msh4 Unc5b Mucl2 Vgf Mugl Mug1 Vmn1r90 Mybl2 Vwc21 Myh15 Wfikkn2 Nek10 Wnt11 Neurod6 Neurod6 Wnt6 Nr1h5 Zeb2os Olfr1042 Zfp608 Olfr1043 Olfr1043 Zfp976 Olfr1082 Olfr1090 Olfr1124 Olfr1167 Olfr1205 Olfr1205

Olfr1206 Olfr1223 Olfr1223

Olfr1263 Olfr1263 Olfr1264 Olfr1269 Olfr127 Olfr127 Olfr1291-ps1

Olfr1406 Olfr1469 Olfr215 Olfr273 Olfr328

Olfr355

Olfr372 Olfr372 Olfr390 Olfr427 Olfr456

Olfr466

Olfr481 Olfr522

Olfr6 Olfr601

Olfr603

Olfr706 Olfr706

Olfr727 Olfr727

Olfr728 Olfr728 Olfr741

Olfr801

Olfr812 Olfr812

Olfr816 Olfr816 Olfr822

Olfr860 Olfr890 Olfr923

Olfr943

Otogl Otogl Pi15 Pi15

Pkhd1 Pkhd111 Platr6 Platr6

Pou3f4 Prr9

Pvalb Rhag Sav1 Serpinb9b Skint1 Skint3

Skint5

Slc10a5 Slc10a5 Slc6a4

Smok2a Tcaf3

Tomm201 Trcgl Trcg1 Trdn Ugt1a6a Usp171a Usp17la Vmn1r178 Vmn1r179

Vmn1r33 Vmn1r74 Vmn1r87 Vmn2r102 Vmn2r113 Vmn2r17 Vmn2r52 Vmn2r66 Vmn2r68 Vmn2r76 Vmn2r78 Wnt16 Wnt16

Methods Mouse Lines

[00448] C57BL6/J wild type mice are purchased from Jackson Laboratory(000664) for

optic nerve crush and glaucoma model experiment. For ageing experiment, females from NIA

Aged Rodent Colonies (https://www.nia.nih.gov/research/dab/aged-rodent-colonies- (https://www.nia.nih.gov/research/dab/aged- rodent-colonies-

handbook) are used. Colla1-tetOP-OKS-mCherry/Rosa26-M2rtTA Colla1-tetOP-OKS-mCherry/ Rosa26-M2rtTAalleles allelesare aredescribed describedin in

Bar-Nur et al., Nat Methods, 2014. 11(11): p. 1170-6. All animal work was approved by

Harvard Medical School, Boston Children's Hospital, Mass Eye and Ear Institutional animal

care and use committees.

Production of AAVs

[00449] Vectors

[00449] Vectors of of AAV-TRE-OSK AAV-TRE-OSK were were made made by by cloning cloning mouse mouse Oct4, Oct4, Sox2 Sox2 andand Klf4 Klf4

cDNA into an AAV plasmid consisting of the a Tet Response Element (TRE3G promoter)

and SV40 element. The other vectors were directly chemically synthesized. All pAAVs, as

listed inTable listed in Table6, 6, were were thenthen packaged packaged into ofAAVs into AAVs of serotype serotype 2/2(titers: 2/2 or 2/9 or 2/9 > (titers: 5 X 10¹² >5x 1012

genome copies per milliliter). Adeno associated viruses were produced by Boston Children's

Hospital Viral Core.

Systemical delivery of AAV9 to internal organs

[00450] Expression in internal organs was achieved through retro-orbital injection of

AAV9 (3x1011 TRE-OSK (3x10 TRE-OSK plus plus 7x1011 7x10¹¹ UBC-rtTA4). UBC-rtTA4). 1mg/mL 1mg/mL doxycycline doxycycline was was treated treated 3 3

weeks post injection continuously to induce OSK expression.

WO wo 2020/069373 PCT/US2019/053545

Cell culture and differentiation

[00451] Ear Ear fibroblasts (EFs) fibroblasts werewere (EFs) isolated fromfrom isolated Reprogramming 4F (Jackson Reprogramming Laboratory 4F (Jackson Laboratory

011011) or 3F (Hochedlinger lab) mice and cultured at 37 °C in DMEM (Invitrogen)

containing Gluta- MAX, non-essential amino acids, and 10% fetal bovine serum (FBS). EFs

of WT 4F and WT 3F mice were passaged to P3 and treated with doxycycline (2 mg/ml) for

the indicated time periods in the culture medium.

[00452] SH-SY5Y neuroblastoma cells were obtained from the American Tissue Culture

Collection (ATCC, CRL-2266) and maintained according to ATCC recommendations.

Basically, the cells were cultured in a 1:1 mixture of Eagle's Minimum Essential Medium

(EMEM, ATCC, 30-2003) and F12 medium (ThermoFisher Scientific, 11765054),

supplemented with 10% fetal bovine serum (FBS, Sigma, F0926) and 1 X

penicillin/streptomycin (ThermoFisher Scientific, 15140122). Cells were cultured at 37°C

with with 5% 5% CO2 CO and and 3% 3%O2. O. Cells Cellswere werepassaged whenwhen passaged reaching ~80% ~80% reaching confluency. confluency.

[00453] SH-SY5Y cells were differentiated into neurons as previously described1,2 described1,2,with with

some modifications. Briefly, 1 day after plating, cells started to be differentiated in

EMEM/F12 medium (1:1) containing 2.5% FBS, 1x penicillin/streptomycin, and 10 uM µM all-

trans retinoic acid (ATRA, Stemcell Technologies, 72264) (Differentiation Medium 1) for 3

days, followed by treating the cells in EMEM/F12 (1:1) containing 1% FBS, 1 X

penicillin/streptomycin, and 10 M µMATRA ATRA(Differentiation (DifferentiationMedium Medium2) 2)for for3 3days. days.Cells Cellswere were

then splitted into 35mm cell culture plates coated with poly-D-lysine (ThermoFisher

Scientific, A3890401). 1 day after splitting, neurons were matured in serum-free

BDNF (Alomone labs) (Differentiation Medium 3) for at least 5 days.

Neurite regeneration assay

[00454] TheThe

[00454] differentiated neurons differentiated neurons from fromSH-SY5Y SH-SY5Ycells were cells transduced were with AAV.DJ transduced with AAV.DJ vectors at 106 genomecopy 10 genome copyper percell. cell.55days daysafter aftertransduction, transduction,100 100nM nMvincristine vincristine(Sigma, (Sigma,

V8879) was added to the cells for 24 hours to induce neurite degeneration. After vincristine

treatment, neurons were washed in PBS twice and fresh Differentiation medium 3 was added

back to the plates. Neurons were followed for neurite outgrowth for 2-3 weeks. Phase-

contrast images were taken at 100x magnification every three to four days. Neurite area was

quantified using Image J.

WO wo 2020/069373 PCT/US2019/053545

Cell cycle analysis

[00455] Cells Cells were were harvested harvested and and fixed fixed with with 70% 70% cold cold ethanol ethanol for for 16 16 hours hours at at 4°C. 4°C. After After

fixation, cells were washed twice with PBS, followed by incubation with PBS containing 50

ug/mL µg/mL propidium iodide (Biotium, 40017) and 100 ug/mL µg/mL RNase A (Omega) for 1 hour at

room temperature. PI stained samples were analyzed on BD LSR II analyzer, and only single

cells were gated for analysis. Cell cycle profiles were analyzed using FCS Express 6 (De

Novo Software).

Human neuron methylation studies and epigenetic clock

[00456] DNA was extracted from cells using the Zymo Quick DNA mini-prep plus kit

(D4069) according to the manufacturer's instructions and DNA methylation levels were

measured on Illumina 850 EPIC arrays according to the manufacturer's instructions. The

Illumina BeadChip (EPIC) measures bisulfite-conversion-based, single-CpG resolution

DNAm levels at different CpG sites in the human genome. These data were generated by

following the standard protocol of Illumina methylation assays, which quantifies methylation

levels by levels bythe theß value value using usingthe ratio the of intensities ratio between of intensities methylated between and un-methylated methylated and un-methylated

alleles. Specifically, the value is is ß value calculated from calculated the from intensity the of of intensity the methylated the (M (M methylated

corresponding to signal A) and un-methylated (U corresponding to signal B) alleles, as the

ratio of fluorescent signals = ß Max(M,0)/[Max(M,0)+ Max(U,0)+100]. = Max(M,0)/[Max(M,0)+ Thus, Max(U,0)+100]. B values Thus, range ß values range

from 0 (completely un-methylated) to 1 (com-pletely methylated). We used the "noob"

normalization method, which is implemented in the "minfi" R package (Triche et al., NAR

2013, Fortin et al., Bioinformatics 2017). The mathematical algorithm and available software

underlying the skin & blood clock (based on 391 CpGs) is presented in Horvath et al., Aging

2018.

AAV2 Virus Intravitreal Injection

[00457] For intravitreal injection, adult animals were anesthetized with ketamine/xylazine

(100/10 mg/kg) and then AAV (1-3 ul) µ1) was injected intravitreally, just posterior to the

limbus-parallel conjunctival vessels, with a fine glass pipette attached to the Hamilton

syringe using plastic tubing. In elevated IOP model, mice received a 1,11 intravitrealinjection 1µl intravitreal injection

between 3-4 weeks post microbead injection.

WO wo 2020/069373 PCT/US2019/053545

Optic Nerve Crush

[00458] For optic nerve crush in anesthetized animals, the optic nerve was accessed

intraorbitally and crushed using a pair of Dumont #5 forceps (FST), two weeks after AAV

injection. Alexa- conjugated cholera toxin beta subunit (CTB-555, 1 mg/ml; 1-2 ul) µl) injection

was performed 2-3 days before euthanasia to trace regenerating RGC axons. More detailed

surgical methods were described by Park et al., Science, 2008. 322(5903): p. 963-6.

In Vivo Doxycycline Induction or suppression

[00459] Induction Induction of Tet-On of Tet-On system system or suppression or suppression of Tet-Off of Tet-Off system system in the in the retina retina werewere

performed by administration of doxycycline hyclate(2 mg/ml) (Sigma) in the drinking water.

Induction of Tet-On system in the whole body were performed by administration of

doxycycline (1 mg/ml) (USP grade, MP Biomedicals 0219895505) in the drinking water.

Axon Regeneration Quantification

[00460] Number of regenerating axons in the optic nerve was estimated by counting the

number of CTB-labeled axons at different distances from the crush site as described

previously (Park, K.K., et al., Promoting axon regeneration in the adult CNS by modulation

of the PTEN/mTOR pathway. Science, 2008. 322(5903): p. 963-6).

Whole-Mount Optic Nerve Preparation

Optic

[00461] Optic nerves nerves and and connecting connecting chiasm chiasm werewere dehydrated dehydrated in methanol in methanol for for 5 min, 5 min, thenthen

incubated overnight with Visikol® HISTO-1TM Next day HISTO-1. Next day nerves nerves were were transferred transferred to to

Visikol® HISTO-2TM and HISTO-2 and then then incubated incubated for for 3 3 hr. hr. Finally, Finally, optic optic nerves nerves and and connecting connecting

HISTO-2M. chiasm were mounted with Visikol® HISTO-2TM.

Immunofluorescence

[00462] Whole-mount Whole-mount retinas retinas werewere blocked blocked withwith horse horse serum serum 4 °C4 overnight °C overnight thenthen

incubated at 4 °C for 3 days with primary antibodies: Mouse anti-Oct4 (1:100, BD

bioscience, 611203), Rabbit anti-Sox2 (1:100, Cell signaling, 14962), Goat anti-Klf4 (1:100,

R&D system, AF3158), Rabbit anti-Brn3a (1:200, EMD Millipore, MAB1585), and Guinea

pig anti-RBPMS (1:400, Raygene custom order A008712 to peptide

GGKAEKENTPSEANLQEEEVRC) diluted in PBS, BSA (3%) Triton X-100 (0.5%). Then,

tissues were incubated at 4 °C overnight with appropriate Alexa Fluor conjugate secondary

antibodies (Alexa 405, 488, 567, 674; Invitrogen) diluted with the same blocking solution as the primary antibodies, generally used at 1:400 final dilution. For section staining, primary overnight at 4 °C and then secondary at room temperature for 2 h. Sections or whole-mount retinas were mounted with VECTASHIELD Antifade Mounting Medium.

Western Blot

[00463] SDS-PAGE and western blot analysis was performed according to standard

procedures and detected with the ECL detection kit. Antibody used: Rabbit anti-Sox2 (1:100,

EMD Millipore, AB5603), Mouse anti-Klf4 (1:1000, ReproCell, 09-0021), Rabbit anti-p-S6

(S240/244) (1:1000, CST, 2215), Mouse anti-S6 (1:1000, CST, 2317), Mouse anti-ß-Tubulin

(1:1000, Sigma-Aldrich, 05-661), Mouse anti-B-Actin-Peroxidase anti-}-Actin-Peroxidase antibody (1:20,000,

Sigma-Aldrich, A3854).

RGCs Survival and Phospho-S6 Signal

RBPMS-positive

[00464] RBPMS-positive cells cells in the in the ganglion ganglion layer layer werewere counted counted using using a fluorescent a fluorescent mi- mi-

croscope after immunostaining whole-mount retinas with anti-RBPMs antibodies. A total of

four random fields per retina were enumerated. The average number per field was

determined, and the percentages of viable RGCs were obtained by comparing the values

determined from the uninjured contralateral retinas. In the same condition, after phospho-S6

staining, the densities of phopsho- S6-positive RGCs were obtained by comparing the value

from the uninjured contralateral retinas.

RGC Enrichment

Retinas

[00465] Retinas werewere fresh fresh dissected dissected and and dissociated dissociated in AMES in AMES media media using using papain, papain, thenthen

triturated carefully and stained with Thy1.2-PE-Cy7 anti- body (Invitrogen 25-0902-81) and

Calcine Blue live-dead cell stain, then flow sorted on a BD FACS Aria using an 130um 130µm

nozzle to collect over 10,000 Thy1.2+ and Clacine blue+ cells (1-2% of total events). Freezed

cells were sent to Genewiz for RNA extraction and ultra low input RNA-seq sequencing, or

to Zymo research for DNA extraction and ultra low input RRBS sequencing.

Classic RRBS Library preparation

[00466] DNA was extracted using Quick-DNA Plus Kit Microprep Kit. 2-10 ng of starting

input genomic DNA was digested with 30 units of Mspl MspI (NEB). Fragments were ligated to

pre-annealed adapters containing 5' -methyl-cytosine instead of cytosine according to

WO wo 2020/069373 PCT/US2019/053545

Illumina' S specified guidelines. Adaptor-ligated fragments 50 bp in size were recovered

using the DNA Clean & ConcentratorTM-5 (Cat#: D4003). The fragments were then

bisulfite-treated using the EZ DNA Methylation-LightningTM Kit (Cat#: D5030).

Preparative-scale PCR was performed and the resulting products were purified with DNA

Clean & ConcentratorTM-5 (Cat#: D4003) for sequencing on an Illumina HiSeq using

2x125bp PE.

DNA methylation age analysis of mouse RGC

Reads

[00467] Reads werewere filtered filtered using using trimtrim galore galore v0.4.1 v0.4.1 and and mapped mapped to the to the mouse mouse genome genome

GRCm38 using Bismark v0.15.0. Methylation counts on both positions of each CpG site

were combined. Only CpG sites covered in all samples were considered for analysis. This

resulted in total of 708156 sites. For the rDNA methylation clock reads were mapped to

BK000964 and the coordinates were adjusted accordingly (Wang et al., Genome Res 29, 325-

333, doi:10.1101/gr.241745.118 doi: 10.1101/gr.241745.118(2019)). (2019)).70/72 70/72sites siteswere werecovered coveredfor forrDNA rDNAclock, clock,compared compared

to 102/435 sites of whole lifespan multi-tissue clock (Meer et al., Elife 7,

doi:10.7554/eLife.40675 doi: 10.7554/eLife.40675(2018)), (2018)),or or248/582 248/582and and77,342/ 77,342/193,651 193,651sites sites(ridge) (ridge)of oftwo twoentire entire

lifespan multi-tissue clocks (Thompson et al., Aging (Albany NY) 10, 2832-2854,

doi: 10.18632/aging.101590 10.18632/aging. 101590(2018)). (2018)).

Microbead-induced mouse model of elevated IOP

[00468] Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100

mg/kg; Ketaset; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (9 mg/kg;

TranquiVed; Vedco, Inc., St. Joseph, MO) supplemented by topical application of

proparacaine (0.5%; Bausch & Lomb, Tampa, FL). Elevation of IOP was induced unilaterally

by injection of polystyrene microbeads (FluoSpheres; Invitrogen, Carlsbad, CA; 15-um 15-µm

diameter) to the anterior chamber of the right eye of each animal under a surgical microscope,

as previously reported (Krishnan et al., J Immunol, 2016. 197(12): p. 4626-4638). Briefly,

microbeads were prepared at a concentration of 5.0 x X 106 beads/mL in 10 beads/mL in sterile sterile physiologic physiologic

saline. The right cornea was gently punctured near the center using a sharp glass micropipette

(World Precision Instruments Inc., Sarasota, FL). A 2 uL µL volume of microbeads was injected

through the preformed hole into the anterior chamber followed by injection of an air bubble

via the micropipette connected with a Hamilton syringe. Any mice that developed signs of

inflammation (clouding of the cornea, edematous cornea etc) were excluded from the study.

WO wo 2020/069373 PCT/US2019/053545

IOP (Intraocular pressure) measurements

[00469] IOPs were measured with a rebound TonoLab tonometer (Colonial Medical

Supply, Espoo, Finland), as previously described (Krishnan et al., J Immunol, 2016. 197(12):

p. 4626-4638; Mukai et al., PLoS One, 2019. 14(1): p. e0208713). Mice were anesthetized by

3% isoflurane in 100% oxygen (induction) followed by 1.5% isoflurane in 100% oxygen

(maintenance) delivered with a precision vaporizer. IOP measurement was initiated within 2

to 3 min after the loss of a toe pinch reflex or tail pinch response. Anesthetized mice were

placed on a platform and the tip of the pressure sensor was placed approximately 1/8 inch

from the central cornea. Average IOP was displayed automatically after 6 measurements after

elimination of the highest and lowest values. The machine-generated mean was considered as

one reading, and six readings were obtained for each eye. All IOPs were taken at the same

time of day (between 10:00 and 12:00 hours) due to the variation of IOP throughout the day.

Optomotor Response

[00470] Visual Visual acuity acuity of mice of mice was was measured measured using using an optomotor an optomotor re- re- flex-based flex-based spatial spatial

frequency threshold test (Gao et al., Am J Pathol, 2016. 186(4): p. 985-1005; Sun et al., Glia,

2013. 61(8): p. 1218-1235). Mice would be able to freely move and were placed on a pedestal

located in the center of an area formed by four computer monitors arranged in a quadrangle.

The monitors displayed a moving vertical black and white sinusoidal grating pattern. A

blinded observer, unable to see the direction of the moving bars, monitored the tracking

behavior of the mouse. Tracking was considered positive when there was a movement of the

head (motor response) to the direction of the bars or rotation of the body in the direction

concordant with the stimulus. Each eye would be tested separately depending on the direction

of rotation of the grating. The staircase method was used to determine the spatial frequency

start from 0.15 to 0.40 cycles/deg, the interval is 0.05 cycles/deg. Rotation speed (12 %/s) (12%) andand

contrast (100%) were kept constant. Responses were measured before and after treatment by by

individuals blinded to the group of the animal and the treatment.

Pattern Electroretinogram (pERG)

[00471] MiceMice werewere anesthetized withwith anesthetized ketamine/xylazine (100mg/kg ketamine/xylazine and and (100mg/kg 20mg/kg) and and 20mg/kg) the the

pupils dilated with one drop of 1% tropicamide ophthalmic solution. The mice were placed

on a built-in warming plate (Celeris, Full-Field and Pattern Stimulation for the rodent model),

that maintained the body temperature at 37 C and kept under dim red light throughout the

WO wo 2020/069373 PCT/US2019/053545

procedure. The visual stimuli of a black and white reversing checkerboard pattern with a

check size of 1° was displayed on light guide electrode-stimulators placed directly on the

ocular surface of both eyes and centered with the pupil. The visual stimuli were presented at

98% contrast and constant mean luminance of 50 cd/m², spatial frequency :0.05 cyc/deg;

temporal frequency : 1Hz. A total of 300 complete contrast reversals of pERG were repeated

twice in each eye and the 600 cycles were segmented and averaged and recorded. The

averaged PERGs were analyzed to evaluate the peak to trough N1 to P1 (positive wave)

amplitude.

Quantification of optic nerve axons

[00472] For quantification of axons, optic nerves were dissected and fixed overnight in

Karnovsky's reagent (50% in phosphate buffer). Semi-thin cross-sections of the nerve were

taken at 1.0 mm posterior to the globe and stained with 1% p-phenylenediamine (PPD) for

evaluation by light microscopy. Optic nerve cross sections were imaged at 60x magnification

using a Nikon microscope (Eclipse E800, Nikon, Japan) with the DPController software

(Olympus, Japan) and 6-8 non-overlapping photomicrographs were taken to cover the entire

area of each optic nerve cross-section. Using ImageJ (Version 2.0.0-rc-65/1.51u), a 100 uM µM X X

100 uM µM square was placed on each 60x image and all axons within the square (0.01mm2) (0.01mm²)

were counted using the threshold and analyze particles function in image J as previously

described (Krishnan et al., J Immunol, 2016. 197(12): p. 4626-4638; Mukai et al., PLoS One,

2019. 14(1): p. e0208713; Gao et al., Am J Pathol, 2016. 186(4): p. 985-1005). Damaged

axons stain darkly with PPD and are not counted. The average axon counts in the 6-8 images

were used to calculate the axon density per square millimeter of optic nerve. Individuals

blinded to the experimental groups performed all axon counts.

Quantification of retinal ganglion cells

[00473] For ganglion cell counting, images of whole mount retinas were acquired using a

63x oil immersion objective of the Leica TCS SP5 confocal microscope (Leica

Microsystems). The retinal whole mount was divided into four quadrants and three to four

images (248.53pm (248.53µm by 248.53pm 248.53µm in size) were taken from the midperipheral and peripheral

regions of each quadrant, for a total of twelve to sixteen images per retina. were taken from

the midperipheral and peripheral regions (4 images per quadrant). The images were obtained

as z-stacks (0.5um) (0.5µm) and all Brn3a positive cells in the ganglion cell layer of each image were

WO wo 2020/069373 PCT/US2019/053545

counted manually as previously described (Gao et al., Am J Pathol, 2016. 186(4): p. 985-

1005). Briefly, RGCs were counted using the "Cell Counter" plugin (fiji.sc/Cell_Counter) in

Fiji is Just ImageJ software (ImageJ Fijivversion Fiji,version 2.0.0-rc-69/1.52n). Each image was loaded

into Fiji and a color counter type was chosen to mark all Brn3a stained RGCs within each

(0.025mm ²).The image (0.025mm²). Theaverage averagenumber numberof ofRGCs RGCsin inthe the12 12to tosixteen sixteenimages imageswere wereused usedto to

calculate the RGC density per square millimeter of retina. Two individuals blinded to the

experimental groups performed all RGC counts.

Total RNA extraction and Sample QC

[00474] Total RNA was extracted following the Trizol Reagent User Guide (Thermo

Fisher Scientific). 1 ul 10 mg/ml Glycogen was added to the supernatant to increase RNA

recovery. RNA was quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad,

CA, USA) and RNA integrity was checked with TapeStation (Agilent Technologies, Palo

Alto, CA, USA) to see if the concentration met the requirements.

Ultra-low input RNA library preparation and multiplexing

[00475] RNA RNA samples samples werewere quantified quantified using using Qubit Qubit 2.0 2.0 Fluorometer Fluorometer (Life (Life Technologies, Technologies,

Carlsbad, CA, USA) and RNA integrity was checked with 2100 TapeStation (Agilent

Technologies, Palo Alto, CA, USA). RNA library preparations, sequencing reactions, and

initial bioinformatics analysis were conducted at GENEWIZ, LLC. (South Plainfield, NJ,

USA). SMART-Seq v4 Ultra Low Input Kit for Sequencing was used for full-length cDNA

synthesis and amplification (Clontech, Mountain View, CA), and Illumina Nextera XT

library was used for sequencing library preparation. Briefly, cDNA was fragmented and

adaptor was added using Transposase, followed by limited-cycle PCR to enrich and add

index to the cDNA fragments. The final library was assessed with Qubit 2.0 Fluorometer and

Agilent TapeStation.

Sequencing 2x150bp PE

[00476] The The sequencing sequencing libraries libraries werewere multiplexed multiplexed and and clustered clustered on two on two lanes lanes of aof a

flowcell. After clustering, the flowcell were loaded on the Illumina HiSeq instrument

according to manufacturer's instructions. The samples were sequenced using a 2x150 Paired

End (PE) configuration. Image analysis and base calling were conducted by the HiSeq

Control Software (HCS) on the HiSeq instrument. Raw sequence data (.bcl files) generated

WO wo 2020/069373 PCT/US2019/053545

from Illumina HiSeq were be converted into fastq files and de-multiplexed using Illumina

bcl2fastq V. 2.17 program. One mis-match was allowed for index sequence identification.

RNA-seq analysis

Paired-end

[00477] Paired-end reads reads werewere aligned aligned withwith hisat2 hisat2 v2.1.0 v2.1.0 to the to the Ensembl Ensembl GRCm38 GRCm38

primary assembly using splice junctions from the Ensembl release 84 annotation. Paired read

counts were quantified using featureCounts v1.6.4 using reads with a MAPQ ==20. >=20.

Diffentially-expressed genes for each pairwise comparison were identified with edgeR v3.26,

testing only genes with at least 0.1 counts-per-million (CPM) in at least three samples. Gene

ontology analysis of differentially-expressed genes was performed with AmiGO v2.5.12.

Age-associated sensory perception genes were extracted from the mouse Sensory Perception

(GO:0007600) category the Gene Ontology database, including genes that were differentially

expressed (q<=0.05) in 12 versus 5 month old mice, excluding genes that were induced by

the the Control Controlvirus alone virus (q<=0.1). alone (q<=0.1).

Table 6. AAV vectors used in Example 16

Vector qPCR Primer for measuring titer Source

pAAV-TRE-Oct4 TRE3G Disclosed herein

pAAV-TRE-Sox2 Disclosed herein TRE3G pAAV-TRE-KIf4 Disclosed herein TRE3G pAAV-TRE-Oct4-Sox2 Disclosed herein TRE3G pAAV-TRE-OSK Disclosed herein TRE3G pAAV-TRE-d2EGFP Disclosed herein TRE3G pAAV-CMV-rtTAV16 Disclosed herein WPRE WPRE pAAV-CAG-ITA pAAV-CAG-tTA Disclosed herein hGH hGH pAAV-sh-Scr-YFP WPRE WPRE Plasmid #85741 pAAV-Sh-Tet1-YFP WPRE Plasmid #85742 WPRE pAAV-sh-Tet2-YFP WPRE Plasmid #85743 WPRE

Table 7. Primers

Primer name Sequence SEQ ID NO: TRE3G F 53 AACGTATCTACAGTTTACTCCCTATC TRE3G R 54 GGTAGGAAGTGGTACGGAAAG GGTAGGAAGTGGTACGGAAAG 55 WPRE F CACTGACAATTCCGTGGTG CACTGACAATTCCGTGGTGT 56 WPRE R GAGATCCGACTCGTCTGAGG hGH F 57 TGGGAAGACAACCTGTAGGC TGGGAAGACAACCTGTAGGG hGH R 58 TGAAACCCCGTCTCTACCAA

Table 8. Primers used for RT-PCR

Gene Gene Primer sequence SEQ ID NO: mOct4 F ACA TCG CCA ATC AGC TTG G 59

mOct4 R AGA ACC ATA CTC GAA CCA CAT CC 60 61 mSox2 F ACA GAT GCA ACC GAT GCA CC mSox2 R TGG AGT TGT ACT GCA GGG CG 62

mKlf4 F 63 GTGCCCCGACTAACCGTTG GTGCCCCGACTAACCGTTG mKlf4 mKlf4 RR 64 GTCGTTGAACTCCTCGGTCT mMyc F 65 ATGCCCCTCAACGTGAACTTC mMyc R 66 CGCAACATAGGATGGAGAGCA CGCAACATAGGATGGAGAGCA mHist1 h2a F GCG ACA ACA AGA AGA CGC GCA T 67

mHist1 h2a R CTG GAT GTT GGG CAG GAC GCC 68

mHist1 h2b F AAG AAG GAC GGC AAG AAG CGC A 69

mHist1 h2b R CGC TCG AAG ATG TCG TTC ACG A 70 70 mHIST1 H3.1/H3.2 F 71 GAA GAA GCC TCA CCG CTA CCG mHIST1 H3.1/H3.2 R GGT TGG TGT CCT CAA ACA GAC CC 72 72 mHist1 h4 F AAC ATC CAG GGC ATC ACC AAG C 73

mHist1 h4 R GTT CTC CAG GAA CAC CTT CAG C 74

mLmnb1 F CCG GCC TCA AGG CTC TCT A 75

mLmnb1 R TGC CGC CTC ATA CTC TCG AA 76

mActb FF mActb AGT GTG ACG TTG ACA TCC GT 77

mActb R TGC TAG GAG CCA GAG CAG TA 78

mNanog F 79 TCTTCCTGGTCCCCACAGTTT mNanog R 80 GCAAGAATAGTTCTCGGGATGAA 81 81 mChafla R GTG TCT TCC TCA ACT TTC TCC TTG G mChafla F CGC GGA CAG CCG CGG CCG TGG ATT GC 82

mChaf1b R GGC TCC TTG CTG TCA TTC ATC TTC CAC 83

mChaf1b FF mChaf1b CAC CGC CGT CAG GAT CTG GAA GTT GG 84 85 85 mLmnb1 F CCG GCC TCA AGG CTC TCT A mLmnb1 R TGC CGC CTC ATA CTC TCG AA 86

mTet1 F 87 TCAAGCAATGGACCACTGGG mTet1 R 88 TCTCCATGAGCTCCCTGACA mTet2 F ACT CCT GGT GAA CAA AGT CAG A 89

mTet2 RR mTet2 CAT CCC TGA GAG CTC TTG CC 90 90 CCA ATG TGT CCG TCG TGG ATC T 91 mGAPDH F GTT GAA GTC GCA GGA GAC AAC C 92 mGAPDH R mp16 (Cdkn2a) F ACA TCA AGA CAT CGT GCG ATA TT 93

mp16 (Cdkn2a) R CCA GCG GTA CAC AAA GAC CA 94

mApob F AAG CAC CTC CGA AAG TAC GTG 95

mApob R CTC CAG CTC TAC CTT ACA GTT GA 96 96 hTet2 F GATAGAACCAACCATGTTGAGGG 97 hTet2 R 98 TGGAGCTTTGTAGCCAGAGGT

182 wo WO 2020/069373 PCT/US2019/053545 hActb F 99 CACCATTGGCAATGAGCGGTTC hActb R 100 AGGTCTTTGCGGATGTCCACGT

Example 17. Non-limiting examples of sequences.

[00478] Nucleotide sequence encoding Mus Musculus OCT4 (no stop codon) (SEQ ID

NO: 1):

ATGGCTGGACACCTGGCTTCAGACTTCGCCTTCTCACCCCCACCAGGTGGGGGTG ATGGGTCAGCAGGGCTGGAGCCGGGCTGGGTGGATCCTCGAACCTGGCTAAGO ATGGGTCAGCAGGGCTGGAGCCGGGCTGGGTGGATCCTCGAACCTGGCTAAGCT TCCAAGGGCCTCCAGGTGGGCCTGGAATCGGACCAGGCTCAGAGGTATTGGGGA TCTCCCCATGTCCGCCCGCATACGAGTTCTGCGGAGGGATGGCATACTGTGGACC TCTCCCCATGTCCGCCCGCATACGAGTTCTGCGGAGGGATGGCATACTGTGGACC TCAGGTTGGACTGGGCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAG CAGGTTGGACTGGGCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAG GGCCAGGCAGGAGCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCC GGCCAGGCAGGAGCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCO TGTGCCGACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGA0 TGTGCCGACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGAG GAGTCCCAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTG GAGTCCCAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTG CTGAAGCAGAAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCAC< CTGAAGCAGAAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCACC CTGGGCGTTCTCTTTGGAAAGGTGTTCAGCCAGACCACCATCTGTCGCTTCGAGG CTGGGCGTTCTCTTTGGAAAGGTGTTCAGCCAGACCACCATCTGTCGCTTCGAGG CCTTGCAGCTCAGCCTTAAGAACATGTGTAAGCTGCGGCCCCTGCTGGAGAAGTG CCTTGCAGCTCAGCCTTAAGAACATGTGTAAGCTGCGGCCCCTGCTGGAGAAGTG GGTGGAGGAAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAGA GGTGGAGGAAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAGA CCCTGGTGCAGGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGO CCCTGGTGCAGGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGT GGAGTCTGGAGACCATGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCA GGAGTCTGGAGACCATGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCAC ACATCGCCAATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTC TCACATCGCCAATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTCTGT AACCGGCGCCAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAACGAGAAGAG ATGAGGCTACAGGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCC TATGAGGCTACAGGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCCC CAGGTCCCCACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTA CAGGTCCCCACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTA CTCAGTCCCTTTTCCTGAGGGCGAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTG CTCAGTCCCTTTTCCTGAGGGCGAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTGG GCTCTCCCATGCATTCAAAC GCTCTCCCATGCATTCAAAC

[00479] Amino

[00479] Amino acid acid sequence sequence encoding encoding MusMus Musculus Musculus OCTOCT 4 (SEQ 4 (SEQ ID ID NO:NO: 2):2):

MAGHLASDFAFSPPPGGGDGSAGLEPGWVDPRTWLSFQGPPGGPGIGPGSEVLGISP MAGHLASDFAFSPPPGGGDGSAGLEPGWVDPRTWLSFQGPPGGPGIGPGSEVLGISE CPPAYEFCGGMAYCGPQVGLGLVPQVGVETLQPEGQAGARVESNSEGTSSEPCADR CPPAYEFCGGMAYCGPQVGLGLVPQVGVETLQPEGQAGARVESNSEGTSSEPCADR PNAVKLEKVEPTPEESQDMKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFG PNAVKLEKVEPTPEESQDMKALQKELEQFAKLLKQKRITLGYTQADVGLTLGVLFG KVFSQTTICRFEALQLSLKNMCKLRPLLEKWVEEADNNENLQEICKSETLVQARKRK KVFSQTTICRFEALQLSLKNMCKLRPLLEKWVEEADNNENLQEICKSETLVQARKRK RTSIENRVRWSLETMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRSSI RTSIENRVRWSLETMFLKCPKPSLQQITHIANQLGLEKDVVRVWFCNRRQKGKRSS

183

EYSQREEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGEAFPSVP) EYSQREEYEATGTPFPGGAVSFPLPPGPHFGTPGYGSPHFTTLYSVPFPEGEAFPSVPV TALGSPMHSN

[00480] Nucleotide sequence encoding Mus Musculus SOX2 (no stop codon) (SEQ ID

NO: 3):

ATGTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCG T'GTATAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTT GGGGGCGGCGGCGGAGGAGGCAACGCCACGGCGGCGGCGACCGGCGGCAA0 GGGGGCGGCGGCGGAGGAGGCAACGCCACGGCGGCGGCGACCGGCGGCAACCA GAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGGTATGGTC GAAGAACAGCCCGGACCGCGTCAAGAGGCCCATGAACGCCTTCATGGTATGGTC CCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGO CCGGGGGCAGCGGCGTAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGG AGATCAGCAAGCGCCTGGGCGCGGAGTGGAAACTTTTGTCCGAGACCGAGAAGO AGATCAGCAAGCGCCTGGGCGCGGAGTGGAAACTTTTGTCCGAGACCGAGAAGC GGCCGTTCATCGACGAGGCCAAGCGGCTGCGCGCTCTGCACATGAAGGAGCACC GGCCGTTCATCGACGAGGCCAAGCGGCTGCGCGCTCTGCACATGAAGGAGCACC CGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATA CGGATTATAAATACCGGCCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATA AGTACACGCTTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCG AGTACACGCTTCCCGGAGGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCG GGGTTGGGGTGGGCGCCGGCCTGGGTGCGGGCGTGAACCAGCGCATGGACAGCT GGGTTGGGGTGGGCGCCGGCCTGGGTGCGGGCGTGAACCAGCGCATGGACAGCT ACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAG ACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAG TGGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCA CTGGGCTACCCGCAGCACCCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAA CCGATGCACCGCTACGACGTCAGCGCCCTGCAGTACAACTCCATGACCAGCTCG0 CCGATGCACCGCTACGACGTCAGCGCCCTGCAGTACAACTCCATGACCAGCTCGC AGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCA AGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCA CCCCCGGTATGGCGCTGGGCTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTC CAGCCCCCCCGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGG CAGCCCCCCCGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGG ACCTCCGGGACATGATCAGCATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCC GCTGCGCCCAGTAGACTGCACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCC GCTGCGCCCAGTAGACTGCACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCC GGCACGGCCATTAACGGCACACTGCCCCTGTCGCACATO GGCACGGCCATTAACGGCACACTGCCCCTGTCGCACATG

[00481] Amino acid sequence encoding Mus Musculus SOX2 (translated) (SEQ ID NO:

4)

(NMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFM MYNMMETELKPPGPQQASGGGGGGGNATAAATGGNQKNSPDRVKRPMNAFMVW SRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEH SRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPD KYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSY YKYRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYA HMNGWSNGSYSMMQEQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQ TYMNGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDL RDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGTAINGTLPLSHM RDMISMYLPGAEVPEPAAPSRLHMAQHYQSGPVPGTAINGTLPLSHM

184

[00482] Nucleotide sequence encoding Mus Musculus KLF4 (no stop codon) (SEQ ID

NO: 5): NO: 5):

ATGAGGCAGCCACCTGGCGAGTCTGACATGGCTGTCAGCGACGCTCTGCTCCCGT CCTTCTCCACGTTCGCGTCCGGCCCGGCGGGAAGGGAGAAGACACTGCGTCCA CCTTCTCCACGTTCGCGTCCGGCCCGGCGGGAAGGGAGAAGACACTGCGTCCAG CAGGTGCCCCGACTAACCGTTGGCGTGAGGAACTCTCTCACATGAAGCGACTTCC CAGGTGCCCCGACTAACCGTTGGCGTGAGGAACTCTCTCACATGAAGCGACTTCC CCACTTCCCGGCCGCCCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGO CCCACTTCCCGGCCGCCCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGGA GAGTGGCGGAGCTGGTGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTAGCCCC GAGTGGCGGAGCTGGTGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTAGCCCG GAGGGAGACCGAGGAGTTCAACGACCTCCTGGACCTAGACTTTATCCTTTCCAAC GAGGGAGACCGAGGAGTTCAACGACCTCCTGGACCTAGACTTTATCCTTTCCAAC TCGCTAACCCACCAGGAATCGGTGGCCGCCACCGTGACCACCTCGGCGTCAGCTT TCGCTAACCCACCAGGAATCGGTGGCCGCCACCGTGACCACCTCGGCGTCAGCTT CATCCTCGTCTTCCCCAGCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAG CTTCAGCTATCCGATCCGGGCCGGGGGTGACCCGGGCGTGGCTGCCAGCAACAC GGTGGAGGGCTCCTCTACAGCCGAGAATCTGCGCCACCTCCCACGGCCCCC AGGTGGAGGGCTCCTCTACAGCCGAGAATCTGCGCCACCTCCCACGGCCCCCTTC AACCTGGCGGACATCAATGACGTGAGCCCCTCGGGCGGCTTCGTGGCTGAGCTC CTGCGGCCGGAGTTGGACCCAGTATACATTCCGCCACAGCAGCCTCAGCCGCCA CTGCGGCCGGAGTTGGACCCAGTATACATTCCGCCACAGCAGCCTCAGCCGCCA GGTGGCGGGCTGATGGGCAAGTTTGTGCTGAAGGCGTCTCTGACCACCCCTGGCA GGTGGCGGGCTGATGGGCAAGTTTGTGCTGAAGGCGTCTCTGACCACCCCTGGCA GCGAGTACAGCAGCCCTTCGGTCATCAGTGTTAGCAAAGGAAGCCCAGACGGCA GCCACCCCGTGGTAGTGGCGCCCTACAGCGGTGGCCCGCCGCGCATGTGCCCCA AGATTAAGCAAGAGGCGGTCCCGTCCTGCACGGTCAGCCGGTCCCTAGAGGCCC AGATTAAGCAAGAGGCGGTCCCGTCCTGCACGGTCAGCCGGTCCCTAGAGGCCC ATTTGAGCGCTGGACCCCAGCTCAGCAACGGCCACCGGCCCAACACACACGACT ATTTGAGCGCTGGACCCCAGCTCAGCAACGGCCACCGGCCCAACACACACGACT TCCCCCTGGGGCGGCAGCTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGC TCCCCCTGGGGCGGCAGCTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGG ACTGCTGAACAGCAGGGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTO AACTGCTGAACAGCAGGGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTCCA TCCCCATCCGGGGCCCAACTACCCTCCTTTCCTGCCAGACCAGATGCAGTCACAA TCCCCATCCGGGGCCCAACTACCCTCCTTTCCTGCCAGACCAGATGCAGTCACAA GTCCCCTCTCTCCATTATCAAGAGCTCATGCCACCGGGTTCCTGCCTGCCAGAGG GTCCCCTCTCTCCATTATCAAGAGCTCATGCCACCGGGTTCCTGCCTGCCAGAGG AGCCCAAGCCAAAGAGGGGAAGAAGGTCGTGGCCCCGGAAAAGAACAGCCACC AGCCCAAGCCAAAGAGGGGAAGAAGGTCGTGGCCCCGGAAAAGAACAGCCACC CACACTTGTGACTATGCAGGCTGTGGCAAAACCTATACCAAGAGTTCTCATCTCA CACACTTGTGACTATGCAGGCTGTGGCAAAACCTATACCAAGAGTTCTCATCTCA AGGCACACCTGCGAACTCACACAGGCGAGAAACCTTACCACTGTGACTGGGACG AGGCACACCTGCGAACTCACACAGGCGAGAAACCTTACCACTGTGACTGGGACG GCTGTGGGTGGAAATTCGCCCGCTCCGATGAACTGACCAGGCACTACCGCAAAC ACACAGGGCACCGGCCCTTTCAGTGCCAGAAGTGCGACAGGGCCTTTTCCAGGT ACACAGGGCACCGGCCCTTTCAGTGCCAGAAGTGCGACAGGGCCTTTTCCAGGT CGGACCACCTTGCCTTACACATGAAGAGGCAC CGGACCACCTTGCCTTACACATGAAGAGGCAC

[00483] Amino acid sequence encoding Mus Musculus KLF4 (translated) (SEQ ID NO:

6):

MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRPAGAPTNRWREELSHMKRLPP) PGRPYDLAATVATDLESGGAGAACSSNNPALLARRETEEFNDLLDLDFILSNSLTHOE PGRPYDLAATVATDLESGGAGAACSSNNPALLARRETEEFNDLLDLDFILSNSLTHQE wo WO 2020/069373 PCT/US2019/053545

SVAATVTTSASASSSSSPASSGPASAPSTCSFSYPIRAGGDPGVAASNTGGGLLYSRES APPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKA APPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASL TPGSEYSSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEA TTPGSEYSSPSVISVSKGSPDGSHPVVVAPYSGGPPRMCPKIKQEAVPSCTVSRSLEAH LSAGPQLSNGHRPNTHDFPLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGP LSAGPQLSNGHRPNTHDFPLGRQLPTRTTPTLSPEELLNSRDCHPGLPLPPGFHPHPGP NYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKPKRGRRSWPRKRTATHTCDYAC NYPPFLPDQMQSQVPSLHYQELMPPGSCLPEEPKPKRGRRSWPRKRTATHTCDYAG CGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFO CGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQ CQKCDRAFSRSDHLALHMKRH

TRE3G

[00484] TRE3G

[00484] promoter sequence promoter sequence (non-limiting (non-limitingexample of a example ofTRE promoter) a TRE (SEQ ID promoter) (SEQ ID

NO: 7):

TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGAT AGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT AGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATA TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATA GAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTT GAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTT ACTCCCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCT ATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAA ATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAA CACTTTTGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAA CACTTTTGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAA

[00485] SV40-derivedterminator

[00485] SV40-derived terminator sequence sequence(SEQ (SEQIDID NO:NO: 8):8):

TGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTTATAATGGTTA TGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTTATAATGGTTAC AAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCAT AAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATI CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCTCGGTA CCG

[00486]

[00486] T2Asequence T2A sequence(SEQ (SEQIDIDNO: NO:9): 9):GSGEGRGSLLTCGDVEENPGP GSGEGRGSLLTCGDVEENPGP

[00487] Nucleotide sequence encoding rtTA3(with 2 VP16 domain at 3' end) (SEQ ID

NO: 10):

ATGTCTAGGCTGGACAAGAGCAAAGTCATAAACGGAGCTCTGGAATTACTCAAT ATGTCTAGGCTGGACAAGAGCAAAGTCATAAACGGAGCTCTGGAATTACTCAAT GGTGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTT GTGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTT GAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCC GAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCC TGCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGO CTGCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCG AGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCT AGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTCCT CTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACA GTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTC GTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTG GAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTAT GGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCAC TGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACC GATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGA GATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAG CCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACA GCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAG GCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGA CATGCTCCCAGCCGATGCCCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAA CATGCTCCCAGCCGATGCCCTTGACGATTTTGACCTTGACATGCTCCCCGGGTAA

[00488] Amino

[00488] Amino acidsequence acid sequence encoding encoding rtTA3 rtTA3(SEQ ID ID (SEQ NO:NO: 11): 11):

ISRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALP MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALP IEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYET LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPL LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDD LRQAIELFDRQGAEPAFLFGLELICGLEKQLKCESGGPTDALDDFDLDMLPADALDD FDLDMLPG

[00489] Nucleotide sequence encoding rtTA4 (with 3 VP16 domain at 3' end) (SEQ ID

NO: 12):

ATGTCCCGCTTGGATAAGAGCAAGGTAATAAATAGCGCACTCGAACTCCTCAAC ATGTCCCGCTTGGATAAGAGCAAGGTAATAAATAGCGCACTCGAACTCCTCAAC GGCGTGGGCATCGAAGGTCTGACTACTCGAAAGCTCGCCCAGAAATTGGGTGTC GGCGTGGGCATCGAAGGTCTGACTACTCGAAAGCTCGCCCAGAAATTGGGTGTG AGCAACCTACATTGTATTGGCATGTCAAGAACAAAAGAGCCCTGCTGGACGCT GAGCAACCTACATTGTATTGGCATGTCAAGAACAAAAGAGCCCTGCTGGACGCT CTTCCTATTGAAATGCTTGACAGGCATCACACTCATTCCTGCCCCCTTGAGGTC< CTTCCTATTGAAATGCTTGACAGGCATCACACTCATTCCTGCCCCCTTGAGGTCG AGAGTTGGCAAGATTTTCTCCGAAACAATGCAAAGTCCTACCGCTGCGCACTTTT AGAGTTGGCAAGATTTTCTCCGAAACAATGCAAAGTCCTACCGCTGCGCACTTTT GTCCCATAGGGATGGAGCAAAAGTGCACCTGGGAACCAGGCCAACAGAGAAAC GTCCCATAGGGATGGAGCAAAAGTGCACCTGGGAACCAGGCCAACAGAGAAAC AATACGAGACTCTCGAGAACCAGTTGGCTTTCTTGTGCCAACAGGGGTTCTCACT GAAAATGCCCTTTACGCACTGTCAGCCGTTGGACATTTTACCCTGGGGTGCGT TGAAAATGCCCTTTACGCACTGTCAGCCGTTGGACATTTTACCCTGGGGTGCGTT CTTGAGGAGCAAGAACATCAGGTTGCTAAGGAGGAGCGCGAGACTCCAACCAC CTTGAGGAGCAAGAACATCAGGTTGCTAAGGAGGAGCGCGAGACTCCAACCACT GATTCTATGCCACCTTTGCTGAAACAGGCCATTGAACTTTTCGATAGACAGGGTG GATTCTATGCCACCTTTGCTGAAACAGGCCATTGAACTTTTCGATAGACAGGGTG CTGAACCTGCCTTTCTCTTCGGGTTGGAGCTGATTATTTGTGGTCTCGAAAAACA CTGAACCTGCCTTTCTCTTCGGGTTGGAGCTGATTATTTGTGGTCTCGAAAAACA GCTGAAATGTGAAAGTGGTGGCCCTACTGACGCCCTCGATGATTTCGACCTGGAT GCTGAAATGTGAAAGTGGTGGCCCTACTGACGCCCTCGATGATTTCGACCTGGAT ATGCTGCCAGCCGATGCACTTGATGATTTCGATTTGGATATGCTTCCAGCCGACG ATGCTGCCAGCCGATGCACTTGATGATTTCGATTTGGATATGCTTCCAGCCGACG CACTGGACGACTTCGATTTGGACATGCTTCCCGGTTAA CACTGGACGACTTCGATTTGGACATOCTTCCCGGTTAA

[00490] Amino acid sequence encoding rtTA4 (SEQ ID NO: 13):

MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPI MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALPI 187

MLDRHHTHSCPLEVESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYE7 EMLDRHHTHSCPLEVESWQDFLRNNAKSYRCALLSHRDGAKVHLGTRPTEKQYETL NQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPP ENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL QAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPADALDDP KQAIELFDRQGAEPAFLFGLELICGLEKQLKCESGGPTDALDDFDLDMLPADALDDF DLDMLPADALDDFDLDMLPG

[00491] Nucleotide

[00491] Nucleotidesequence sequence encoding encoding M2-rtTA M2-rtTA(SEQ ID ID (SEQ NO:NO: 14):14):

ATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTA ATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTAT AAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG AAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG GCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCAC CACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGG CACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGG AACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCAC TGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCI TGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCT GTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTC AATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCC AATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGC GTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAT GTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAT CGATACCGTCGACCTCGAGACCTAGAAAAACATGGAGCAATCACAAGTAGCAAT ACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAG- ACAGCAGCTACCAATGCTGATTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAG GTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAG TGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAAT CTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTC ACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTA ACTCCCAACGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTA CTTCCCTGATTGGCAGAACTACACACCAGGGCCAGGGATCAGATATCCACTGAC CTTCCCTGATTGGCAGAACTACACACCAGGGCCAGGGATCAGATATCCACTGAC CTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCAAGAGAAGGTAGAAGAAGC CAATGAAGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGA CAATGAAGGAGAGAACACCCGCTTGTTACACCCTGTGAGCCTGCATGGGATGGA TGACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCA TGACCCGGAGAGAGAAGTATTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCA TCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTAGACCAG TCACATGGCCCGAGAGCTGCATCCGGACTGTACTGGGTCTCTCTGGTTAGACCAG ATCTGA

[00492]

[00492] Amino acid Amino sequence acid encoding sequence M2-rtTA encoding (SEQ M2-rtTA ID ID (SEQ NO:NO: 15): 15):

MPLYHAIASRMAFIFSSLYKSWLLSLYEELWPVVRQRGVVCTVFADATPTGWGIAT7 COLLSGTFAFPLPIATAELIAACLARCWTGARLLGTDNSVVLSGKSSSFPWLLACVAT WILRGTSFCYVPSALNPADLPSRGLLPALRPLPRLRLRPOTSRISLWAASPHRYRRPR EKHGAITSSNTAATNADCAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHP KEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGIRYPLTFGWCYKLVP KEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGIRYPLTFGWCYKLVPV wo WO 2020/069373 PCT/US2019/053545

EQEKVEEANEGENTRLLHPVSLHGMDDPEREVLEWRFDSRLAFHHMARELHPDCTO SLWLDQI

[00493] Nucleic

[00493] Nucleic acid acid sequence sequence of of pAAV-TRE3G-OSK-SV40pA, pAAV-TRE3G-OSK-SV40pA, TRE-OSK-SV40, TRE-OSK-SV40, or or

TRE3G-OSK-SV40pA vector (SEQ ID NO: 16):

TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTG TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG/ CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTAT ACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTAT AACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGG TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT CAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTT TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTO CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCG TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCG CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAAC CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCT ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTT ATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCC AATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCG TCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGC AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGC ACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACTCCCTATCA TACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACTCCCTATCAGT GATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGC GATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAACGTATGCAG ACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTG ACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAG ATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGTATCTACAG "TACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGA' TTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTATCAGTGATA GAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCC GAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCG TTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGTCTTAT AACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCGCCACCATGGCTGGAC AACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCGCCACCATGGCTGGAC ACCTGGCTTCAGACTTCGCCTTCTCACCCCCACCAGGTGGGGGTGATGGGTCAGO ACCTGGCTTCAGACTTCGCCTTCTCACCCCCACCAGGTGGGGGTGATGGGTCAGC AGGGCTGGAGCCGGGCTGGGTGGATCCTCGAACCTGGCTAAGCTTCCAAGGGCC AGGGCTGGAGCCGGGCTGGGTGGATCCTCGAACCTGGCTAAGCTTCCAAGGGCC TCCAGGTGGGCCTGGAATCGGACCAGGCTCAGAGGTATTGGGGATCTCCCCATGT TCCAGGTGGGCCTGGAATCGGACCAGGCTCAGAGGTATTGGGGATCTCCCCATGT CCGCCCGCATACGAGTTCTGCGGAGGGATGGCATACTGTGGACCTCAGGTTGGA CTGGGCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAGGGCCAGGCA CTGGGCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAGGGCCAGGCA GAGCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCCTGTGCCGA GGAGCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCCTGTGCCGAC CGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGAGGAGTCCCAG CGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGAGGAGTCCCAG BACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTGCTGAAGO GACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTGCTGAAGCAG AAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCACCCTGGGCGTT AAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCACCCTGGGCGTT CTTTGGAAAGGTGTTCAGCCAGACCACCATCTGTCGCTTCGAGGCCTTGC CTCTTTGGAAAGGTGTTCAGCCAGACCACCATCTGTCGCTTCGAGGCCTTGCAGC TCAGCCTTAAGAACATGTGTAAGCTGCGGCCCCTGCTGGAGAAGTGGGTGGAGC TCAGCCTTAAGAACATGTGTAAGCTGCGGCCCCTGCTGGAGAAGTGGGTGGAGG AAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAGACCCTGGTGC AAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAGACCCTGGTGC GGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGTGGAGTCTG AGGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGTGGAGTCTGG AGACCATGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCACTCACATCGO AGACCATGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCACTCACATCGC AATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTCTGTAACCGGO CAATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTCTGTAACCGGCGO CAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAACGAGAAGAGTATGAGO CAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAACGAGAAGAGTATGAGGCT ACAGGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCCCCAGGTCCCC ACAGGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCCCCAGGTCCCC ACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTACTCAGTCCC ACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTACTCAGTCCC TTCCTGAGGGCGAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTGGGCTCTCC TTTTCCTGAGGGCGAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTGGGCTCTCCCA TGCATTCAAACGCTAGCGGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGC TGCATTCAAACGCTAGCGGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGC AGGAGATGTTGAAGAAAACCCCGGGCCTGCATGCATGTATAACATGATGGAGAC AGGAGATGTTGAAGAAAACCCCGGGCCTGCATGCATGTATAACATGATGGAGAG

GGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAG GGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAGGAG GCAACGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCC GCAACGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGACCGC GTCAAGAGGCCCATGAACGCCTTCATGGTATGGTCCCGGGGGCAGCGGCGTAAG GTCAAGAGGCCCATGAACGCCTTCATGGTATGGTCCCGGGGGCAGCGGCGTAAG ATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTGGGC ATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTGGGC GCGGAGTGGAAACTTTTGTCCGAGACCGAGAAGCGGCCGTTCATCGACGAGGCC AAGCGGCTGCGCGCTCTGCACATGAAGGAGCACCCGGATTATAAATACCGGCCC AAGCGGCTGCGCGCTCTGCACATGAAGGAGCACCCGGATTATAAATACCGGCCG GGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGCTTCCCGGAGGC CGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGCTTCCCGGAGGC TTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGC TTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCCGGC CTGGGTGCGGGCGTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGG CTGGGTGCGGGCGTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGCTGG AGCAACGGCAGCTACAGCATGATGCAGGAGCAGCTGGGCTACCCGCAGCACCCG GGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGACGTC GGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGACGTC GCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGC" AGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGCTCG CCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCCGGTATGGCGCTGGGCT CCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCCGGTATGGCGCTGGGCT CCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGTTACCTC TTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAG TTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATCAGC ATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCAC ATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTGCAC ATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACGGCCATTAACGGCACA ATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACGGCCATTAACGGCACA CTGCCCCTGTCGCACATGGCATGCGGCTCCGGCGAGGGCAGGGGAAGTCTTCTA CTGCCCCTGTCGCACATGGCATGCGGCTCCGGCGAGGGCAGGGGAAGTCTTCTA CATGCGGGGACGTGGAGGAAAATCCCGGCCCACTCGAGATGAGGCAGCCA0 ACATGCGGGGACGTGGAGGAAAATCCCGGCCCACTCGAGATGAGGCAGCCACCT GGCGAGTCTGACATGGCTGTCAGCGACGCTCTGCTCCCGTCCTTCTCCACGTTCG CGTCCGGCCCGGCGGGAAGGGAGAAGACACTGCGTCCAGCAGGTGCCCCGACT CGTCCGGCCCGGCGGGAAGGGAGAAGACACTGCGTCCAGCAGGTGCCCCGACTA ACCGTTGGCGTGAGGAACTCTCTCACATGAAGCGACTTCCCCCACTTCCCGGCC ACCGTTGGCGTGAGGAACTCTCTCACATGAAGCGACTTCCCCCACTTCCCGGCCG CCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGGAGAGTGGCGGAGCTC CCCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGGAGAGTGGCGGAGCTGG TGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTAGCCCGGAGGGAGACCGAGGA TGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTAGCCCGGAGGGAGACCGAGGA GTTCAACGACCTCCTGGACCTAGACTTTATCCTTTCCAACTCGCTAACCCACCA GTTCAACGACCTCCTGGACCTAGACTTTATCCTTTCCAACTCGCTAACCCACCAG GAATCGGTGGCCGCCACCGTGACCACCTCGGCGTCAGCTTCATCCTCGTCTTCC GAATCGGTGGCCGCCACCGTGACCACCTCGGCGTCAGCTTCATCCTCGTCTTCCC AGCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCAGCTATCCGAT CAGCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCAGCTATCCGAT CCGGGCCGGGGGTGACCCGGGCGTGGCTGCCAGCAACACAGGTGGAGGGCTCCT CCGGGCCGGGGGTGACCCGGGCGTGGCTGCCAGCAACACAGGTGGAGGGCTCCT TACAGCCGAGAATCTGCGCCACCTCCCACGGCCCCCTTCAACCTGGCGGACATO CTACAGCCGAGAATCTGCGCCACCTCCCACGGCCCCCTTCAACCTGGCGGACATC AATGACGTGAGCCCCTCGGGCGGCTTCGTGGCTGAGCTCCTGCGGCCGGAGTTGC AATGACGTGAGCCCCTCGGGCGGCTTCGTGGCTGAGCTCCTGCGGCCGGAGTTGG ACCCAGTATACATTCCGCCACAGCAGCCTCAGCCGCCAGGTGGCGGGCTGATGO ACCCAGTATACATTCCGCCACAGCAGCCTCAGCCGCCAGGTGGCGGGCTGATGG CAAGTTTGTGCTGAAGGCGTCTCTGACCACCCCTGGCAGCGAGTACAGCA0 GCAAGTTTGTGCTGAAGGCGTCTCTGACCACCCCTGGCAGCGAGTACAGCAGCC CTTCGGTCATCAGTGTTAGCAAAGGAAGCCCAGACGGCAGCCACCCCGTGGTAG GGCGCCCTACAGCGGTGGCCCGCCGCGCATGTGCCCCAAGATTAAGCAAGAGG TGGCGCCCTACAGCGGTGGCCCGCCGCGCATGTGCCCCAAGATTAAGCAAGAGG

CGGTCCCGTCCTGCACGGTCAGCCGGTCCCTAGAGGCCCATTTGAGCGCTGGACC CCAGCTCAGCAACGGCCACCGGCCCAACACACACGACTTCCCCCTGGGGCGGC CCAGCTCAGCAACGGCCACCGGCCCAACACACACGACTTCCCCCTGGGGCGGCA CTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGGAACTGCTGAACAGCAG GCTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGGAACTGCTGAACAGCAG GGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTCCATCCCCATCCGGGGCCC GGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTCCATCCCCATCCGGGGCCC AACTACCCTCCTTTCCTGCCAGACCAGATGCAGTCACAAGTCCCCTCTCTCCATT TCAAGAGCTCATGCCACCGGGTTCCTGCCTGCCAGAGGAGCCCAAGCCAAA ATCAAGAGCTCATGCCACCGGGTTCCTGCCTGCCAGAGGAGCCCAAGCCAAAGA GGGGAAGAAGGTCGTGGCCCCGGAAAAGAACAGCCACCCACACTTGTGACTATO GGGGAAGAAGGTCGTGGCCCCGGAAAAGAACAGCCACCCACACTTGTGACTATG AGGCTGTGGCAAAACCTATACCAAGAGTTCTCATCTCAAGGCACACCTGCGAA CAGGCTGTGGCAAAACCTATACCAAGAGTTCTCATCTCAAGGCACACCTGCGAA CTCACACAGGCGAGAAACCTTACCACTGTGACTGGGACGGCTGTGGGTGGAAAT CTCACACAGGCGAGAAACCTTACCACTGTGACTGGGACGGCTGTGGGTGGAAAT CGCCCGCTCCGATGAACTGACCAGGCACTACCGCAAACACACAGGGCACCGGC TCGCCCGCTCCGATGAACTGACCAGGCACTACCGCAAACACACAGGGCACCGGC CCTTTCAGTGCCAGAAGTGCGACAGGGCCTTTTCCAGGTCGGACCACCTTGCCTT CACATGAAGAGGCACTAAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAA ACACATGAAGAGGCACTAAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAA CTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTO CTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTC ACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAA GTATCTTATCATGTCTGGATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTT TGTATCTTATCATGTCTGGATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTT CTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTA CCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAA GGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT GGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT GAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA GAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCA TGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTT GTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTAC AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACA AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACA CCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTT TCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCC CAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTA CAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTA AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTT CCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTA CCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTAC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATC GGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATC GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAG7 GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG ACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGA GACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGAT TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAAC AAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCAT CTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA CTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA TATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGC AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGG CATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATG CATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGC 192 wo WO 2020/069373 PCT/US2019/053545

TGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGT TGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGT AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTT. AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTA AAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAA0 AAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACA GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAA GAAAAGCATCTTACGGATGGCATGACAGTAAGAGAA

[00494] Nucleic acid sequence of AAV-UBC-rtTA4-WPRE3-SV40pA pAAV-UBC-rtTA4-WPRE3-SV40pAvector vector(SEQ (SEQID ID

NO: 17): NO:17):

TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATO CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG CGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAAC' ACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTAT TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA AACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATT GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACO AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC TTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACA CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCT TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACO TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG

WO 2020/069373 wo PCT/US2019/053545

TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCC TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCG CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCA GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTT ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTT ATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG AATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCG CGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGC AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGC AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGC ACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCCTGATCTGGCCTCCC TACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCCTGATCTGGCCTCCG CGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCA CGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGA CGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGA CAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGG CAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGG ACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAG ACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAG CGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCC CGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCC GTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGO GTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCT AGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGA AGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGAT CGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGG GCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGG GCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGG CCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATO TCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCAGCAAAATGGC GGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGTGAGGTCGT7 GGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGGGCTGTGAGGTCGTT GAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTC GAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTC GCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGG CCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGC ACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTGTCGTCTGTTGCGG GGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCG GGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCG GCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGG CGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGG CCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCC CCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCG GGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAG GGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTA GCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGA GCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAA GTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTO GTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTG TTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGACC TTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGACG AGCGGCCGCATTAAACGCCACCATGTCCCGCTTGGATAAGAGCAAGGTAAT AAGCGGCCGCATTAAACGCCACCATGTCCCGCTTGGATAAGAGCAAGGTAATAA ATAGCGCACTCGAACTCCTCAACGGCGTGGGCATCGAAGGTCTGACTACTCGA ATAGCGCACTCGAACTCCTCAACGGCGTGGGCATCGAAGGTCTGACTACTCGAA AGCTCGCCCAGAAATTGGGTGTGGAGCAACCTACATTGTATTGGCATGTCAAGA

ACAAAAGAGCCCTGCTGGACGCTCTTCCTATTGAAATGCTTGACAGGCATCACAL ACAAAAGAGCCCTGCTGGACGCTCTTCCTATTGAAATGCTTGACAGGCATCACAC TCATTCCTGCCCCCTTGAGGTCGAGAGTTGGCAAGATTTTCTCCGAAACAATGC TCATTCCTGCCCCCTTGAGGTCGAGAGTTGGCAAGATTTTCTCCGAAACAATGCA AAGTCCTACCGCTGCGCACTTTTGTCCCATAGGGATGGAGCAAAAGTGCACCTGG AAGTCCTACCGCTGCGCACTTTTGTCCCATAGGGATGGAGCAAAAGTGCACCTGG AACCAGGCCAACAGAGAAACAATACGAGACTCTCGAGAACCAGTTGGCTTTC GAACCAGGCCAACAGAGAAACAATACGAGACTCTCGAGAACCAGTTGGCTTTCT GTGCCAACAGGGGTTCTCACTTGAAAATGCCCTTTACGCACTGTCAGCCGTTGG TGTGCCAACAGGGGTTCTCACTTGAAAATGCCCTTTACGCACTGTCAGCCGTTGG ACATTTTACCCTGGGGTGCGTTCTTGAGGAGCAAGAACATCAGGTTGCTAAGGAG ACATTTTACCCTGGGGTGCGTTCTTGAGGAGCAAGAACATCAGGTTGCTAAGGAG GAGCGCGAGACTCCAACCACTGATTCTATGCCACCTTTGCTGAAACAGGCCATTC GAGCGCGAGACTCCAACCACTGATTCTATGCCACCTTTGCTGAAACAGGCCATTG ACTTTTCGATAGACAGGGTGCTGAACCTGCCTTTCTCTTCGGGTTGGAGCT AACTTTTCGATAGACAGGGTGCTGAACCTGCCTTTCTCTTCGGGTTGGAGCTGAT TATTTGTGGTCTCGAAAAACAGCTGAAATGTGAAAGTGGTGGCCCTACTGACGCC TCGATGATTTCGACCTGGATATGCTGCCAGCCGATGCACTTGATGATTTCGA' CTCGATGATTTCGACCTGGATATGCTGCCAGCCGATGCACTTGATGATTTCGATTT GGATATGCTTCCAGCCGACGCACTGGACGACTTCGATTTGGACATGCTTCCCGGT GGATATGCTTCCAGCCGACGCACTGGACGACTTCGATTTGGACATGCTTCCCGGT AAACTAGTCTAGCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGG TAAACTAGTCTAGCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGG TATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTT TATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTT GTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTG GTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTG GTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGAG GTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACA GGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTC GGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTG ATGCTATTGCTTTATTTGTAACCATTCTAGCTTTATTTGTGAAATTTGTGATGCTA TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTG TTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTG CATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGG CATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGG GGATCCAAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGC GGATCCAAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGC ATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTC ATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCC CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGG CTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATT CCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAA CCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTT CCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTA CCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCG CCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGA AGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG AGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG GGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAC GGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAG GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCG CGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTT CCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT CCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCT TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGG TTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGG GTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA GTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAG GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTO AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATA TGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATA

TTAACGTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTGCGCGGAACCCCTA TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCO TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCC GATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT TGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCC GTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAG AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT AAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT ACATCGAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA ACATCGAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA ACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC ACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATO GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAG7 ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT- AAGAGAA

[00495]

[00495] UBCUBC promoter promoter sequence sequence (SEQ (SEQ ID ID NO:NO: 18): 18):

GATCTGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCAC GATCTGGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACG GCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGC GCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCCGCC CGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAC CGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAG TATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTT TATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTT TCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGC TCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGC GGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGC GGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGG TGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTG" TGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTG GATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTT GATCGCTGTGATCGTCACTTGGTGAGTAGCGGGCTGCTGGGCTGGCCGGGGCTTT CGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAG CGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGG GCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAG GCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGC CAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCC GCAGCAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTGAGGCGG GCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAA0 GCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAG GTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGG GTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGG GCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTC GCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGTTTG TCGTCTGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACC TCGTCTGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACC TTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATA. TTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAAT GCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGA GCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGA CGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCT CGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCT GGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTA GGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATCTACCTA CTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGT TCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGT GTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATO GTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATG

196 wo WO 2020/069373 PCT/US2019/053545

TAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTT TAATTTTCAGTGTTAGACTAGTAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTT TTTTGTTAGAC

[00496]

[00496] Tet-O Tet-Osequence sequence(SEQ (SEQIDIDNO: NO:19): 19):TCCCTATCAGTGATAGAGA TCCCTATCAGTGATAGAGA

[00497] Nucleicacid

[00497] Nucleic acidsequence sequence encoding encodingminimal minimalCMV promoter CMV (SEQ(SEQ promoter ID NO: ID 20): NO: 20):

GCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGT GCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGT CAGATCGCCTGGA

[00498] Nucleic

[00498] Nucleicacid acidsequence sequence encoding encodingWPRE WPRE(SEQ ID ID (SEQ NO:NO: 21):21):

[00499]

[00499] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCA AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCA TGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGT TCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGC TCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT CGGCTGTTGGGCACTGACAATTCCGTGGTGTT CGGCTGTTGGGCACTGACAATTCCGTGGTGTT

[00500] Nucleic acid sequence encoding inverted terminal repeat sequence (SEQ ID NO:

22):

CCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG CCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGG GCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GGGAGTGGCCAACTCCATCACTAGGGGTTCCT

Nucleic

[00501] Nucleic acidacid sequence sequence of aofTRE2 a TRE2 promoter promoter (a non-limiting (a non-limiting example example of aofTRE a TRE

promoter) (SEQ ID NO: 23):

AATTCGTACACGCCTACCTCGACCCATCAAGTGCCACCTGACGTCTCCCTATCAC AATTCGTACACGCCTACCTCGACCCATCAAGTGCCACCTGACGTCTCCCTATCAG TGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGT CTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATO CTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATC AGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGA AGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGA ACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTA CACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACCTCTCCCTA TCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGT ACCCCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCC ACCCCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCC ATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGGATC< ATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGGATCG C

197 wo WO 2020/069373 PCT/US2019/053545

Nucleic

[00502] Nucleic acidacid sequence sequence of Poftight P tight promoter promoter (a non-limiting (a non-limiting example example of aofTRE a TRE

promoter) (SEQ ID NO: 24):

GAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGT GAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAG GATAGAGAACGATGTCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGTCGA GTTTACTCCCTATCAGTGATAGAGAACGTATGTCGAGTTTACTCCCTATCAGTGA AGAGAACGTATGTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTT TAGAGAACGTATGTCGAGTTTATCCCTATCAGTGATAGAGAACGTATGTCGAGTT TACTCCCTATCAGTGATAGAGAACGTATGTCGAGGTAGGCGTGTACGGTGGGAG GCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCC GCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCC

[00503] Nucleic

[00503] Nucleicacid acidsequence sequence encoding encodingTetR TetR(SEQ ID ID (SEQ NO:NO: 25):25):

ATGGCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAAT ATGGCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAAT GAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGT GAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCC GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGA TAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGA AAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTA AAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTA AGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAG CATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTA TATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAG AGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTAT AGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATT GGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTG GGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACT ATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGC ATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGC AGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAA AGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAA CTTAAATGTGAAAGTGGG

[00504] Amino acid sequence encoding TetR (SEQ ID NO: 26):

MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALA MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALA (EMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYET IEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYET LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPL LRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG LRQAIELFDHQGAEPAFLFGLELICGLEKQLKCESG

[00505] Nucleic

[00505] Nucleicacid acidsequence sequence encoding encodingTetR-Krab TetR-Krab(SEQ ID NO: (SEQ 27) 27) ID NO:

ATGGCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAAT ATGGCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAAT GAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGA TAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGA AAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTA AAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTA AGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAG TATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAG TATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAG AGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTAT AGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATT GGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTG GAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACT ATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGC ATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGC GAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAA AGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAA CTTAAATGTGAAAGTGGGTCGCCAAAAAAGAAGAGAAAGGTCGACGGCGGTGGT CTTAAATGTGAAAGTGGGTCGCCAAAAAAGAAGAGAAAGGTCGACGGCGGTGGT GCTTTGTCTCCTCAGCACTCTGCTGTCACTCAAGGAAGTATCATCAAGAACAAGO GCTTTGTCTCCTCAGCACTCTGCTGTCACTCAAGGAAGTATCATCAAGAACAAGG AGGGCATGGATGCTAAGTCACTAACTGCCTGGTCCCGGACACTGGTGACCTTCA AGGGCATGGATGCTAAGTCACTAACTGCCTGGTCCCGGACACTGGTGACCTTCAA GGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCA GGATGTATTTGTGGACTTCACCAGGGAGGAGTGGAAGCTGCTGGACACTGCTCA CAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCC GCAGATCGTGTACAGAAATGTGATGCTGGAGAACTATAAGAACCTGGTTTCCTTG GGTTATCAGCTTACTAAGCCAGATGTGATCCTCCGGTTGGAGAAGGGAGAAGAG CCTGGCTGGTGGAGAGAGAAATTCACCAAGAGACCCATCCTGATTCAGAGAC CCCTGGCTGGTGGAGAGAGAAATTCACCAAGAGACCCATCCTGATTCAGAGACT GCATTTGAAATCAAATCATCAGTTTAA

[00506]

[00506] Amino acid Amino sequence acid encoding sequence TetR-KRAB encoding (SEQ TetR-KRAB ID ID (SEQ NO:NO: 28): 28):

MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALA MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALA EMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQY IEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYET LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPL LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSPKKKRKVDGGGALSPQH LRQAIELFDHQGAEPAFLFGLELICGLEKQLKCESGSPKKKRKVDGGGALSPQHSAV TQGSIIKNKEGMDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLE NYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSS NYKNLVSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV

[00507]

[00507] Desmin promoter Desmin (SEQ promoter ID ID (SEQ NO:NO: 29): 29):

ACCTTGCTTCCTAGCTGGGCCTTTCCTTCTCCTCTATAAATACCAGCTCTGGTATT ACCTTGCTTCCTAGCTGGGCCTTTCCTTCTCCTCTATAAATACCAGCTCTGGTATT TCGCCTTGGCAGCTGTTGCTGCTAGGGAGACGGCTGGCTTGACATGCATCTCCTG TCGCCTTGGCAGCTGTTGCTGCTAGGGAGACGGCTGGCTTGACATGCATCTCCTO CAAAACACAAACCCGTGGTGTGAGTGGGTGTGGGCGGTGTGAGTAGGGGGAT ACAAAACACAAACCCGTGGTGTGAGTGGGTGTGGGCGGTGTGAGTAGGGGGATG AATCAGAGAGGGGGCGAGGGAGACAGGGGCGCAGGAGTCAGGCAAAGGCGAT AATCAGAGAGGGGGCGAGGGAGACAGGGGCGCAGGAGTCAGGCAAAGGCGATG CGGGGGTGCGACTACACGCAGTTGGAAACAGTCGTCAGAAGATTCTGGAAACTA CGGGGGTGCGACTACACGCAGTTGGAAACAGTCGTCAGAAGATTCTGGAAACTA TCTTGCTGGCTATAAACTTGAGGGAAGCAGAAGGCCAACATTCCTCCCAAGGGA ACTGAGGCTCAGAGTTAAAACCCAGGTATCAGTGATATGCATGTGCCCCGGCC AACTGAGGCTCAGAGTTAAAACCCAGGTATCAGTGATATGCATGTGCCCCGGCC AGGGTCACTCTCTGACTAACCGGTACCTACCCTACAGGCCTACCTAGAGACTCTT TGAAAGGATGGTAGAGACCTGTCCGGGCTTTGCCCACAGTCGTTGGAAACCTC TTGAAAGGATGGTAGAGACCTGTCCGGGCTTTGCCCACAGTCGTTGGAAACCTCA

WO wo 2020/069373 PCT/US2019/053545

GCATTTTCTAGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCATT GCATTTTCTAGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCATT CCAATAACCTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAAACGAAA CCAATAACCTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAAACGAAA TTCTCTAGCCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGGAAAAAAAGGG TTCTCTAGCCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGGAAAAAAAGGG GCCGGCCGGGGGTCTCCTGTCAGCTCCTTGCCCTGTGAAACCCAGCAGGCCTGCC GCCGGCCGGGGGTCTCCTGTCAGCTCCTTGCCCTGTGAAACCCAGCAGGCCTGCC TGTCTTCTGTCCTCTTGGGGCTGTCCAGGGGCGCAGGCCTCTTGCGGGGGAGCTG TGTCTTCTGTCCTCTTGGGGCTGTCCAGGGGCGCAGGCCTCTTGCGGGGGAGCTG CCTCCCCGCCCCCTCGCCTGTGGCCGCCCTTTTCCTGGCAGGACAGAGGGATO GCCTCCCCGCCCCCTCGCCTGTGGCCGCCCTTTTCCTGGCAGGACAGAGGGATCC TGCAGCTGTCAGGGGAGGGGCGCCGGGGGGTGATGTCAGGAGGGCTACAAATAC TGCAGCTGTCAGGGGAGGGGCGCCGGGGGGTGATGTCAGGAGGGCTACAAATAG TGCAGACAGCTAAGGGGCTCCGTCACCCATCTTCACATCCACTCCAGCCGGCTGC CCGCCCGCTGCCTCCTCTGTGCGTCCGCCCAGCCAGCCTCGTCCACGCC CCGCCCGCTGCCTCCTCTGTGCGTCCGCCCAGCCAGCCTCGTCCACGCC

[00508] Desmin-rtTA4vector

[00508] Desmin-rtTA4 vector (SEQ (SEQ ID ID NO: NO:30): 30):

TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTG TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACO ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG CGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTAT ACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTAT TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC GGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT PAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTO GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG AGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGO GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA

TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCG CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGC CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCC/ GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTT ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTT AATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCG AATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCG TCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGC TCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGG AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGC AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGC TACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCCTAGATCTACCTTGC TACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCCTAGATCTACCTTGC TTCCTAGCTGGGCCTTTCCTTCTCCTCTATAAATACCAGCTCTGGTATTTCGCCTT TTCCTAGCTGGGCCTTTCCTTCTCCTCTATAAATACCAGCTCTGGTATTTCGCCTT GGCAGCTGTTGCTGCTAGGGAGACGGCTGGCTTGACATGCATCTCCTGACAAAA GGCAGCTGTTGCTGCTAGGGAGACGGCTGGCTTGACATGCATCTCCTGACAAAAC ACAAACCCGTGGTGTGAGTGGGTGTGGGCGGTGTGAGTAGGGGGATGAATCAGA GAGGGGGCGAGGGAGACAGGGGCGCAGGAGTCAGGCAAAGGCGATGCGGGGGT GAGGGGGCGAGGGAGACAGGGGCGCAGGAGTCAGGCAAAGGCGATGCGGGGGT GCGACTACACGCAGTTGGAAACAGTCGTCAGAAGATTCTGGAAACTATCTTGCTG GCGACTACACGCAGTTGGAAACAGTCGTCAGAAGATTCTGGAAACTATCTTGCTG GCTATAAACTTGAGGGAAGCAGAAGGCCAACATTCCTCCCAAGGGAAACTGAGG GCTATAAACTTGAGGGAAGCAGAAGGCCAACATTCCTCCCAAGGGAAACTGAGG `CAGAGTTAAAACCCAGGTATCAGTGATATGCATGTGCCCCGGCCAGGGTCA CTCAGAGTTAAAACCCAGGTATCAGTGATATGCATGTGCCCCGGCCAGGGTCACT CTCTGACTAACCGGTACCTACCCTACAGGCCTACCTAGAGACTCTTTTGAAAGGA CTCTGACTAACCGGTACCTACCCTACAGGCCTACCTAGAGACTCTTTTGAAAGGA TGGTAGAGACCTGTCCGGGCTTTGCCCACAGTCGTTGGAAACCTCAGCATTTTC TGGTAGAGACCTGTCCGGGCTTTGCCCACAGTCGTTGGAAACCTCAGCATTTTCT AGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCATTCCAATAAC AGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCATTCCAATAAC CTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAAACGAAATTCTCTAC CTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAAACGAAATTCTCTAG CCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGGAAAAAAAGGGGCCGGCCG CCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGGAAAAAAAGGGGCCGGCCG GGGTCTCCTGTCAGCTCCTTGCCCTGTGAAACCCAGCAGGCCTGCCTGTCTT GGGGTCTCCTGTCAGCTCCTTGCCCTGTGAAACCCAGCAGGCCTGCCTGTCTTCT GTCCTCTTGGGGCTGTCCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCC GTCCTCTTGGGGCTGTCCAGGGGCGCAGGCCTCTTGCGGGGGAGCTGGCCTCCCC GCCCCCTCGCCTGTGGCCGCCCTTTTCCTGGCAGGACAGAGGGATCCTGCAGCTC GCCCCCTCGCCTGTGGCCGCCCTTTTCCTGGCAGGACAGAGGGATCCTGCAGCTG TCAGGGGAGGGGCGCCGGGGGGTGATGTCAGGAGGGCTACAAATAGTGCAGAC TCAGGGGAGGGGCGCCGGGGGGTGATGTCAGGAGGGCTACAAATAGTGCAGAG AGCTAAGGGGCTCCGTCACCCATCTTCACATCCACTCCAGCCGGCTGCCCGC AGCTAAGGGGCTCCGTCACCCATCTTCACATCCACTCCAGCCGGCTGCCCGCCCG TGCCTCCTCTGTGCGTCCGCCCAGCCAGCCTCGTCCACGCCAAGCTTGCGGCCG CTGCCTCCTCTGTGCGTCCGCCCAGCCAGCCTCGTCCACGCCAAGCTTGCGGCCG CATTAAACGCCACCATGTCCCGCTTGGATAAGAGCAAGGTAATAAATAGCGCAC CATTAAACGCCACCATGTCCCCCTTCGATAAGAGCAAGGTAATAAATAGCGCAC GAACTCCTCAACGGCGTGGGCATCGAAGGTCTGACTACTCGAAAGCTCG0 AGAAATTGGGTGTGGAGCAACCTACATTGTATTGGCATGTCAAGAACAAAAGAC AGAAATTGGGTGTGGAGCAACCTACATTGTATTGGCATGTCAAGAACAAAAGAG CCCTGCTGGACGCTCTTCCTATTGAAATGCTTGACAGGCATCACACTCATTCCT CCCTGCTGGACGCTCTTCCTATTGAAATGCTTGACAGGCATCACACTCATTCCTGC

CCCCTTGAGGTCGAGAGTTGGCAAGATTTTCTCCGAAACAATGCAAAGTCCTACC CCCCTTGAGGTCGAGAGTTGGCAAGATTTTCTCCGAAACAATGCAAAGTCCTACC GCTGCGCACTTTTGTCCCATAGGGATGGAGCAAAAGTGCACCTGGGAACCAGGO GCTGCGCACTTTTGTCCCATAGGGATGGAGCAAAAGTGCACCTGGGAACCAGGC AACAGAGAAACAATACGAGACTCTCGAGAACCAGTTGGCTTTCTTGTGCCAAG AGGGGTTCTCACTTGAAAATGCCCTTTACGCACTGTCAGCCGTTGGACATTTTAC CCTGGGGTGCGTTCTTGAGGAGCAAGAACATCAGGTTGCTAAGGAGGAGCGCGA CCTGGGGTGCGTTCTTGAGGAGCAAGAACATCAGGTTGCTAAGGAGGAGCGCGA ACTCCAACCACTGATTCTATGCCACCTTTGCTGAAACAGGCCATTGAACTTTTO GACTCCAACCACTGATTCTATGCCACCTTTGCTGAAACAGGCCATTGAACTTTTC BATAGACAGGGTGCTGAACCTGCCTTTCTCTTCGGGTTGGAGCTGATTATTTGTO GATAGACAGGGTGCTGAACCTGCCTTTCTCTTCGGGTTGGAGCTGATTATTTGTG TCTCGAAAAACAGCTGAAATGTGAAAGTGGTGGCCCTACTGACGCCCTCGATG GTCTCGAAAAACAGCTGAAATGTGAAAGTGGTGGCCCTACTGACGCCCTCGATG ATTTCGACCTGGATATGCTGCCAGCCGATGCACTTGATGATTTCGATTTGGATAT ATTTCGACCTGGATATGCTGCCAGCCGATGCACTTGATGATTTCGATTTGGATAT TTCCAGCCGACGCACTGGACGACTTCGATTTGGACATGCTTCCCGGTTAAA GCTTCCAGCCGACGCACTGGACGACTTCGATTTGGACATGCTTCCCGGTTAAACT AGTCTAGCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCT AGTCTAGCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCT AACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATO TAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATC ATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAG ATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAG TCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGC TTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGC TCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTA TCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTA TTGCTTTATTTGTAACCATTCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTT TTGCTTTATTTGTAACCATTCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTI ATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCAT ATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCAT TTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGGGGATCCA TTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGGGGATCCA ATTCCCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGG AATTCCCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCG GTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTC7 GGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGG CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAAT CTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATT TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAAG TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAAC TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGC TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGC CGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACG CCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGC GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAC GCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAC CGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTC TCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAG TCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGG TTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTG GTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGAT GGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGG AGTCCACGTTCTTTAATACTGGACTCTTGTTCCAAACTGGAACAACACTCAACCC AGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACO TCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATT TATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGP TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAAC TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAAC GTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT GTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTGCGCGGAACCCCTATTTC wo WO 2020/069373 PCT/US2019/053545

TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGAT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAA GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATO CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATC GAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT GAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT TCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGT TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT PACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA GAA

[00509] pAAV2_CMV_rtTA(V16) (SEQ ID NO: 31):

AAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGC AAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGC TCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGA/ TCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAAT AGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTA AGCCCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAA GAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC GAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCC ACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAG ACTACGTGAACCATCACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCA CTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCG GCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGC TGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTA GCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAA TGCGCCGCTACAGGGCGCGTACTATGGTTGCTTTGACGTATGCGGTGTGAAATA0 TGCGCCGCTACAGGGCGCGTACTATGGTTGCTTTGACGTATGCGGTGTGAAATAC GCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCCCTGCAGGCAGC CGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCCCTGCAGGCAGCTGC GCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCT GGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA TTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT CCATCACTAGGGGTTCCTGCGGCCGCTCGGTCCGCACGATCTCAATTCGGCCATT CCATCACTAGGGGTTCCTGCGGCCGCTCGGTCCGCACGATCTCAATTCGGCCATT ACGGCCGGATCCGGCTCGAGGAGCTTGGCCCATTGCATACGTTGTATCCATATO ACGGCCGGATCCGGCTCGAGGAGCTTGGCCCATTGCATACGTTGTATCCATATCA TAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGAT TATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA TATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATA ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC TATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCC AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA AACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAA TAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGCTAAACTGCCCACTT GGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA GGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTG0 CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGG CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC

ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCC ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCC AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACO AAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACG GTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGA GTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAG ACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTC ACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTC CGCGGCCCCGAATTCACCATGTCTAGACTGGACAAGAGCAAAATCATAAACAGO CGCGGCCCCGAATTCACCATGTCTAGACTGGACAAGAGCAAAATCATAAACAGC CTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAAC' GCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTC GCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAC GCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAG CGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCA CGGGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCAC AGCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAA0 AGCTGCCCCCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAG CATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCA TCATACCGCTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCA CCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGT CCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGT ITCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCA GTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCA CTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGA CTTTACACTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGA AAGAGAGACACCTACCACCGATTCTATGCCCCCACTTCTGAAGCAAGCAATTGA GCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATO GCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATC ATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGO ATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCGACGCC CTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACO CTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCT TGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGT TGATATGCTGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGT AACTAAGTAAGGATCATCTTAATTAAATCGATAAGGATCTGGCCGCCTCGGCCTA AACTAAGTAAGGATCATCTTAATTAAATCGATAAGGATCTGGCCGCCTCGGCCTA TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATG ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTO TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTG CTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTT CTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTT GAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTT ATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGC GACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG TGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCG GGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCC GGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCG CGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCC CGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCC GCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGAC AGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCAGACATGATAAGATACATTGAT GAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCAGACATGATAAGATACATTGAT GAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAA GAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGA ATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAA ATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAA CAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTT CAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTT TTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAACTAGCGCGTGCGGCCGC TTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAACTAGCGCGTGCGGCCGCAG

GAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACT GAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAG GAGCGAGCGAGCGCGCAGCTGCCTGCAGGACATGTGAGCAAAAGGCCAGCAA TGAGCGAGCGAGCGCGCAGCTGCCTGCAGGACATGTGAGCAAAAGGCCAGCAA AGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCC CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCC TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCO TGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCG TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTAT CGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACT CCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGC AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA AGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATC GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATC TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG TGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCG GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTG GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAA AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA AGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACC ATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTC TATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATA AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTA GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT AATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAG TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAAC GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTC GTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCE ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCC AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA CAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCA CCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAA TCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCG AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGC GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA GCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGA AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTC AAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG CCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA CACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAA ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGT" AACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTT GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT GAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT

CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCAT CGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCAT ACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTT GACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCT GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCT TGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGT GTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGA oGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGA GAGTGCACCATA

[00510]

[00510] CAG-tTA (SEQ CAG-tTA (SEQ ID NO: NO: 32): 32):

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAC GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG GGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGGAGO AGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTGGAGCTA GTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT GTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTT CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC CCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCC CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTCAATAGGGACT CGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTCAATAGGGACTT TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC TCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACA TCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG TCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT CATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACAT ACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACAT AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATT CAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATT GACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCG GACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCG AACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGO TAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGT CTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCA CTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCA CGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCG CGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCG TCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGT AATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGAC GTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAAT GTAAGTACCGCCTATAGAGTCTATAGGCCCACAAAAAATGCTTTCTTCTTTTAAT ATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCA ATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCA AAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGA ATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTGATA ATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATA ATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAA ATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTAC ATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTAC CATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTA GGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGC GGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCA ACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACA ACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATTCGAACA TCGATTGAATTCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGG TCGATTGAATTCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGG AATTACTCAATGAAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAA AATTACTCAATGAAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAA AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCC AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCC GCTCGATGCCCTGGCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCC TGCTCGATGCCCTGGCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCC CCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCO CCTGGAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCG CTGTGCTCTCCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCA CAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCA ACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAA GGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACAG GGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACAG TGGGCTGCGTATTGGAGGATCAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAG TGGGCTGCGTATTGGAGGATCAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAG ACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCC ACACCTACCACCGATTCTATGCCCCCACTTCTGAGACAAGCAATTGAGCTGTTCG ACCATCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGG ACCATCAGGGAGCCGAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGG CCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGGCCGACGCCCTTGACGA CCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGGCCGACGCCCTTGACGA TTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGC TTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGC TGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGATGAGGATO TGCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGATGAGGATC CTCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCAGCGCTGCTCGAGAGATCTAC GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCA GGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCA TCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGAC CTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGAC TAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGG CAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGG CAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGG GTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCO AGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGA TTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCT CAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGG CAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGG TCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGC TCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGG ATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTTTGTAGGTAACCA CGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC CGTGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTC TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCC GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGG GGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGG GCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAC TCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTC GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCC GGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCT CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT AATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA AATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA AAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACC AAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGF TTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAA CTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTG

CCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCG CCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGA ATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTO ATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGC CTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCC TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCG CCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCT CCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCI CCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGAC AAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGO GAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGT TCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG7 TTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGI TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTC GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACO GCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATC GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATT GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG GACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG TTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT IGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG ATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAA. ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAAC ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGAT GGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTG GGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGG TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAC TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAG CACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGA GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC. GTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCAC TGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA TGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTO TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGT AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT AGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCT TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAG TGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC GTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCO TGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCG CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGAT CCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGO AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGC GGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACO GGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCT

ACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCG AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG AAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTTTTGCTCACATGT

[00511] pAAV-Tet-O-OSK-SV40LpA (or pAAV-TRE2-OSK-SV40LpA) (SEQ ID NO: 33):

TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTC TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGA CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC CAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTA ACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTAT TAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGA GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT GGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT TTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGO TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT AAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTA AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT AAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCA AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTO AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTC CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTO GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC GGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACAC CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG CGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA GAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCC CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTT TTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACC

WO wo 2020/069373 PCT/US2019/053545

GCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG CAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCC CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG CGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGG GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAC GCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGG CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA CTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACA TTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCC' ATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAGGCCTT AATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCC AATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCG TCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGG AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGO AGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGC TACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCGTACACGCCTACCTC TACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCGTACACGCCTACCTC GACCCATCAAGTGCCACCTGACGTCTCCCTATCAGTGATAGAGAAGTCGACACGT GACCCATCAAGTGCCACCTGACGTCTCCCTATCAGTGATAGAGAAGTCGACACGT TCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATC CTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGT GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTO GATAGAGAAGTCGACACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACGTC TAGAACGTCTCCCTATCAGTGATAGAGAAGTCGACACGTCTCGAGCTCCCTATCA GTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGAC GTGATAGAGAAGGTACGTCTAGAACGTCTCCCTATCAGTGATAGAGAAGTCGAC CGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCCTATATAAGCAGA ACGTCTCGAGCTCCCTATCAGTGATAGAGAAGGTACCCCCTATATAAGCAGAGCT CGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCC CGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCO ATAGAAGACACCGGGACCGATCCAGCCTGGATCGCGGCCGCGCCACCATGGCTC ATAGAAGACACCGGGACCGATCCAGCCTGGATCGCGGCCGCGCCACCATGGCTG GACACCTGGCTTCAGACTTCGCCTTCTCACCCCCACCAGGTGGGGGTGATGGGTO GACACCTGGCTTCAGACTTCGCCTTCTCACCCCCACCAGGTGGGGGTGATGGGTC AGCAGGGCTGGAGCCGGGCTGGGTGGATCCTCGAACCTGGCTAAGCTTCCAAGO AGCAGGGCTGGAGCCGGGCTGGGTGGATCCTCGAACCTGGCTAAGCTTCCAAGG GCCTCCAGGTGGGCCTGGAATCGGACCAGGCTCAGAGGTATTGGGGATCTCCCC GCCTCCAGGTGGGCCTGGAATCGGACCAGGCTCAGAGGTATTGGGGATCTCCCC ATGTCCGCCCGCATACGAGTTCTGCGGAGGGATGGCATACTGTGGACCTCAGGTT ATGTCCGCCCGCATACGAGTTCTGCGGAGGGATGGCATACTGTGGACCTCAGGTT GGACTGGGCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAGGGCCAG GCAGGAGCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCCTGTGCC GCAGGAGCACGAGTGGAAAGCAACTCAGAGGGAACCTCCTCTGAGCCCTGTGCC GACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGAGGAG GACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACCAACTCCCGAGGAGTCC CAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTGCTGAAC CAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTGCTGAAG CAGAAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCACCCTGGGC CAGAAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCACCCTGGGC TTCTCTTTGGAAAGGTGTTCAGCCAGACCACCATCTGTCGCTTCGAGGCCTT GTTCTCTTTGGAAAGGTGTTCAGCCAGACCACCATCTGTCGCTTCGAGGCCTTGC GCTCAGCCTTAAGAACATGTGTAAGCTGCGGCCCCTGCTGGAGAAGTGGGTGC AGCTCAGCCTTAAGAACATGTCTAAGCTGCGGCCCCTGCTGGAGAAGTGGGTGG AGGAAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAGACCCTGO AGGAAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAGACCCTGG GCAGGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGTGGAG7 TGCAGGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGTGGAGTC GGAGACCATGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCACTCACAT TGGAGACCATGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCACTCACAT CGCCAATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTCTGTAACCGG CGCCAATCAGCTTGGGCTAGAGAAGGATGTGGTTCGAGTATGGTTCTGTAACCGG CGCCAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAACGAGAAGAGTATGAG CGCCAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAACGAGAAGAGTATGAG

GCTACAGGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCCCCAGGTC CCCACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTACTCAG' CCCACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTACTCAGT CCCTTTTCCTGAGGGCGAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTGGGCTC CCCTTTTCCTGAGGGCGAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTGGGCTCTC CCATGCATTCAAACGCTAGCGGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCA CCATGCATTCAAACGCTAGCGGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCA AGCAGGAGATGTTGAAGAAAACCCCGGGCCTGCATGCATGTATAACATGATGGA AGCAGGAGATGTTGAAGAAAACCCCGGGCCTGCATGCATGTATAACATGATGGA BACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAC GACGGAGCTGAAGCCGCCGGGCCCGCAGCAAGCTTCGGGGGGCGGCGGCGGAG GAGGCAACGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGAC GAGGCAACGCCACGGCGGCGGCGACCGGCGGCAACCAGAAGAACAGCCCGGAC CGCGTCAAGAGGCCCATGAACGCCTTCATGGTATGGTCCCGGGGGCAGCGGCGT CGCGTCAAGAGGCCCATGAACGCCTTCATGGTATGGTCCCGGGGGCAGCGGCGT AAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTG AAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCAGCAAGCGCCTG GGCGCGGAGTGGAAACTTTTGTCCGAGACCGAGAAGCGGCCGTTCATCGACGAC GGCGCGGAGTGGAAACTTTTGTCCGAGACCGAGAAGCGGCCGTTCATCGACGAG GCCAAGCGGCTGCGCGCTCTGCACATGAAGGAGCACCCGGATTATAAATACCGG GCCAAGCGGCTGCGCGCTCTGCACATGAAGGAGCACCCGGATTATAAATACCGG CCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGCTTCCCGG CCGCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACACGCTTCCCGGA GGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCC GGCTTGCTGGCCCCCGGCGGGAACAGCATGGCGAGCGGGGTTGGGGTGGGCGCC GGCCTGGGTGCGGGCGTGAACCAGCGCATGGACAGCTACGCGCACATGAACGGC TGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCTGGGCTACCCGCAGCAC TGGAGCAACGGCAGCTACAGCATGATGCAGGAGCAGCTGGGCTACCCGCAGCAC CCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGAC CCGGGCCTCAACGCTCACGGCGCGGCACAGATGCAACCGATGCACCGCTACGAC GTCAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGC GTCAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCTACATGAACGGC TCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCCGGTATGGCGCTGC TCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCCGGTATGGCGCTGG CTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGTT GCTCCATGGGCTCTGTGGTCAAGTCCGAGGCCAGCTCCAGCCCCCCCGTGGTTAC CTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATO CTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTCCGGGACATGATC AGCATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTG AGCATGTACCTCCCCGGCGCCGAGGTGCCGGAGCCCGCTGCGCCCAGTAGACTC CACATGGCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACGGCCATTAACGGC ACACTGCCCCTGTCGCACATGGCATGCGGCTCCGGCGAGGGCAGGGGAAGTCTT ACACTGCCCCTGTCGCACATGGCATGCGGCTCCGGCGAGGGCAGGGGAAGTCTTE CTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCACTCGAGATGAGGCAGCCA CTGGCGAGTCTGACATGGCTGTCAGCGACGCTCTGCTCCCGTCCTTCTCCACG CCTGGCGAGTCTGACATGGCTGTCAGCGACGCTCTGCTCCCGTCCTTCTCCACGT TCGCGTCCGGCCCGGCGGGAAGGGAGAAGACACTGCGTCCAGCAGGTGCCCCGA TCGCGTCCGGCCCGGCGGGAAGGGAGAAGACACTGCGTCCAGCAGGTGCCCCGA CTAACCGTTGGCGTGAGGAACTCTCTCACATGAAGCGACTTCCCCCACTTCCCGG CGCCCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGGAGAGTGGCGGA CCGCCCCTACGACCTGGCGGCGACGGTGGCCACAGACCTGGAGAGTGGCGGAGC GGTGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTAGCCCGGAGGGAGACCG TGGTGCAGCTTGCAGCAGTAACAACCCGGCCCTCCTAGCCCGGAGGGAGACCGA GGAGTTCAACGACCTCCTGGACCTAGACTTTATCCTTTCCAACTCGCTAACCCAC GGAGTTCAACGACCTCCTGGACCTAGACTTTATCCTTTCCAACTCGCTAACCCAC AGGAATCGGTGGCCGCCACCGTGACCACCTCGGCGTCAGCTTCATCCTCGTCTT CAGGAATCGGTGGCCGCCACCGTGACCACCTCGGCGTCAGCTTCATCCTCGTCTT CCAGCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCAGCTAL CCCCAGCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCAGCTATCC GATCCGGGCCGGGGGTGACCCGGGCGTGGCTGCCAGCAACACAGGTGGAGGGCT GATCCGGGCCGGGGGTGACCCGGGCGTGGCTGCCAGCAACACAGGTGGAGGGCT CCTCTACAGCCGAGAATCTGCGCCACCTCCCACGGCCCCCTTCAACCTGGCGGAC

ATCAATGACGTGAGCCCCTCGGGCGGCTTCGTGGCTGAGCTCCTGCGGCCGGAGT TGGACCCAGTATACATTCCGCCACAGCAGCCTCAGCCGCCAGGTGGCGGGCTGA TGGGCAAGTTTGTGCTGAAGGCGTCTCTGACCACCCCTGGCAGCGAGTACAGO TGGGCAAGTTTGTGCTGAAGGCGTCTCTGACCACCCCTGGCAGCGAGTACAGCA CCCTTCGGTCATCAGTGTTAGCAAAGGAAGCCCAGACGGCAGCCACCCCGTGG GCCCTTCGGTCATCAGTGTTAGCAAAGGAAGCCCAGACGGCAGCCACCCCGTGG TAGTGGCGCCCTACAGCGGTGGCCCGCCGCGCATGTGCCCCAAGATTAAGCAA TAGTGGCGCCCTACAGCGGTGGCCCGCCGCGCATGTGCCCCAAGATTAAGCAAG AGGCGGTCCCGTCCTGCACGGTCAGCCGGTCCCTAGAGGCCCATTTGAGCGCTGG AGGCGGTCCCGTCCTGCACGGTCAGCCGGTCCCTAGAGGCCCATTTGAGCGCTGG ACCCCAGCTCAGCAACGGCCACCGGCCCAACACACACGACTTCCCCCTGGGGC ACCCCAGCTCAGCAACGGCCACCGGCCCAACACACACGACTTCCCCCTGGGGCG CAGCTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGGAACTGCTGAACAG GCAGCTCCCCACCAGGACTACCCCTACACTGAGTCCCGAGGAACTGCTGAACAG CAGGGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTCCATCCCCATCCGGGG CAGGGACTGTCACCCTGGCCTGCCTCTTCCCCCAGGATTCCATCCCCATCCGGGG CAACTACCCTCCTTTCCTGCCAGACCAGATGCAGTCACAAGTCCCCTCTG CCCAACTACCCTCCTTTCCTGCCAGACCAGATGCAGTCACAAGTCCCCTCTCTCC ATTATCAAGAGCTCATGCCACCGGGTTCCTGCCTGCCAGAGGAGCCCAAGCCA ATTATCAAGAGCTCATGCCACCGGGTTCCTGCCTGCCAGAGGAGCCCAAGCCAA AGAGGGGAAGAAGGTCGTGGCCCCGGAAAAGAACAGCCACCCACACTTGTGACT AGAGGGGAAGAAGGTCGTGGCCCCGGAAAAGAACAGCCACCCACACTTGTGACT ATGCAGGCTGTGGCAAAACCTATACCAAGAGTTCTCATCTCAAGGCACACCTGCG ATGCAGGCTGTGGCAAAACCTATACCAAGAGTTCTCATCTCAAGGCACACCTGCG ACTCACACAGGCGAGAAACCTTACCACTGTGACTGGGACGGCTGTGGGTGGA AACTCACACAGGCGAGAAACCTTACCACTGTGACTGGGACGGCTGTGGGTGGAA ATTCGCCCGCTCCGATGAACTGACCAGGCACTACCGCAAACACACAGGGCACCG ATTCGCCCGCTCCGATGAACTGACCAGGCACTACCGCAAACACACAGGGCACCG GCCCTTTCAGTGCCAGAAGTGCGACAGGGCCTTTTCCAGGTCGGACCACCTTGC GCCCTTTCAGTGCCAGAAGTGCGACAGGGCCTTTTCCAGGTCGGACCACCTTGCC TTACACATGAAGAGGCACTAAATGACTAGTCTAGCAATCAACCTCTGGATTACAA TTACACATGAAGAGGCACTAAATGACTAGTCTAGCAATCAACCTCTGGATTACAA AATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGT AATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTG ATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCAT7 GATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATT TCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGC TTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGC TGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCO CTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG GTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTCTAGCTTTA GTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTCTAGCTTTA TGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATA TTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAA CAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATG CAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGT GGGAGGTTTTTTAAAGCGGGGGATCCAAATTCCCGATAAGGATCTTCCTAGAGCA GGGAGGTTTTTTAAAGCGGGGGATCCAAATTCCCGATAAGGATCTTCCTAGAGCA TGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCT TGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCT AGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGC AGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGG CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG CGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG CGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCC CGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTG ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTT ACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTT CGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTT CGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTT CGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCC GCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGC GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCC GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCC GCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAA GCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAA

GCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCG GCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCG ACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA ACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA PACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCT' GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGT TCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGG TCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGG GATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTT GATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTT ACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGO AACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGGG GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTAT CCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG AGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGAT CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGATCC CAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGATCC TTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC" TTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCT GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGG GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGC CGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGC ATCTTACGGATGGCATGACAGTAAGAGAA ATCTTACGGATGGCATGACAGTAAGAGAA

VP64,

[00512] VP64, 4 repeats 4 repeats of VP16 of VP16 (SEQ(SEQ ID NO: ID NO: 34) 34) (Non-limiting (Non-limiting example example of aof a

transactivation domain):

AGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGO GAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGC GGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCC TGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCT TGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTG GACATGCTGATTAACTCTAGA

[00513] P65 (SEQ ID NO: 35) (Non-limiting example of a transactivation domain):

AGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGA AGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAA GCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCC GCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCC CACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGO CACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGC AGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCA CAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACO TCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCA ATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGG CCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCA CCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCA CCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGO CCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGC GGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGO TGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGE CGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGA CGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGA CTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCC TCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCC wo WO 2020/069373 PCT/US2019/053545

AGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCC CCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTC GTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAC GTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAG GCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGC GCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGC CGATATGGATTTCTCAGCCTTGCTG

[00514] RTA (SEQ ID NO: 36) (Non-limiting example of a transactivation domain):

CGGGATTCCAGGGAAGGGATGTTTTTGCCGAAGCCTGAGGCCGGCTCCGCTATTA GTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGC CAAAACGAA GTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGC CAAAACGAA TCCGGCCA TCCGGCCA TTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCAG TTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCAC CAACACCAACCGGTCCAGTACATGAGCCAGTCGGGTCACTGACCCCGGCACCAG TCCC TCCC CAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCACCTGTTGG GGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGA GGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGA TACTGTGATTCCCCAGAAGGAA TACTGTGATTCCCCAGAAGGAA GAGGCTGCAA GAGGCTGCAA TCTGTGGCCAAA TCTGTGGCCAAA TGGACCTTTCCCA TCCGCCCCCAAGGGGCCA TCTGGA TGAGCT GACAACCACACTTGAGTCCA TGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCT TGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCT GGATACCTTCCTGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGA C TGTCCA TCTTCGACACA TCTCTGTTT

[00515]

[00515] MPHMPH MS2-P65-HSF1 MS2-P65-HSF1 (SEQ (SEQ ID ID NO:NO: 37)37) (Non-limiting (Non-limiting example example of of a a

transactivation domain):

GCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATGTGA GCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATGTGA AGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAA0 CAGTGGCTCCTTCTAATTTCGCTAATGGGGTGGCAGAGTGGATCAGCTCCAACTC ACGGAGCCAGGCCTACAAGGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAA GAGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGG AGAAAGTATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGG GGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACT CGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATGGAGCTCACI ATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGG ATCCCAATTTTCGCTACCAATTCTGACTOTGAACTCATCGTGAAGGCAATGCAGG GGCTCCTCAAAGACGGTAATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTAT CTACAGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTA CTACAGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTA GCGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGATCCCCTTCAGGGCAGA GCGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGATCCCCTTCAGGGCAGA TCAGCAACCAGGCCCTGGCTCTGGCCCCTAGCTCCGCTCCAGTGCTGGCCCAGAC TATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGCTCCAGCCC TATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGCTCCAGCCCCT

GTGCTGACCCCAGGACCACCCCAGTCACTGAGCGCTCCAGTGCCCAAGTCTACA0 GTGCTGACCCCAGGACCACCCCAGTCACTGAGCGCTCCAGTGCCCAAGTCTACAC AGGCCGGCGAGGGGACTCTGAGTGAAGCTCTGCTGCACCTGCAGTTCGACGCTC TGAGGACCTGGGAGCTCTGCTGGGGAACAGCACCGATCCCGGAGTGTTCAG ATGAGGACCTGGGAGCTCTGCTGGGGAACAGCACCGATCCCGGAGTGTTCACAG AATCTGGCCTCCGTGGACAACTCTGAGTTTCAGCAGCTGCTGAATCAGGGCGTGTC CATGTCTCATAGTACAGCCGAACCAATGCTGATGGAGTACCCCGAAGCCATTACC GGCTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGG CGGCTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGGA ACCAGCGGCCTGCCTAATGGGCTGTCCGGAGATGAAGACTTCTCAAGCATCGCTG ACCAGCGGCCTGCCTAATGGGCTGTCCGGAGATGAAGACTTCTCAAGCATCGCTG ATATGGACTTTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAGGGAGGAGC ATATGGACTTTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAGGGAGGAGG TGGAAGCGGCTTCAGCGTGGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTCC GACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTAT GTGACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCC AAGAGCTCCTGTCTCCCCAGGAGCCCCCCAGGCCTCCCGAGGCAGAGAACAGCA AAGAGCTCCTGTCTCCCCAGGAGCCCCCCAGGCCTCCCGAGGCAGAGAACAGCA GCCCGGATTCAGGGAAGCAGCTGGTGCACTACACAGCGCAGCCGCTGTTCCTGC GCCCGGATTCAGGGAAGCAGCTGGTGCACTACACAGCGCAGCCGCTGTTCCTGC IGGACCCCGGCTCCGTGGACACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGO TGGACCCCGGCTCCGTGGACACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGC TGGGAGAGGGCTCCTACTTCTCCGAAGGGGACGGCTTCGCCGAGGACCCCACCA TCTCCCTGCTGACAGGCTCGGAGCCTCCCAAAGCCAAGGACCCCACTGTCTCC

[00516] OCT4-2A-SOX2-2A-KLF4 (non-limiting example of nucleic acid sequence

encoding human OCT4, human SOX2, and human KLF4, each separated by a 2A peptide)

(SEQ ID (SEQ ID NO: NO:38): 38):

ATGGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAG ATGGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAG GTGATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAA GTGATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAA GCTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCT GCTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCT CTGAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGAT GGCGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGA GGCGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGA ACCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGA GACCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGG GCCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGA GGCCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGA GAAGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAC GAAGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAG AACTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATA AACTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATA CACAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCA CACAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCA AACGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAA0 AACGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAG CTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATC CTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTT AGGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAAC CAGGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAAC CAGTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCC CAGTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCC GAAACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAA

GATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAA GGATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAG CAGCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGC CAGCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGG GACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATO GGACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATG GGAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGC GGAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGC CTTTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCG CTTTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCG CAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAA GCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAA ACCCCGGGCCTGCATGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGG GCCCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCG GCCCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCG GCGGCAACCAGAAAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCA GCGGCAACCAGAAAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCA GGTGTGGTCCCGCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGA TGGTGTGGTCCCGCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGC ACAACTCGGAGATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGA ACAACTCGGAGATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGA GGAGAAGCGGCCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACA CGGAGAAGCGGCCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGA AGGAGCACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGA AGGAGCACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGA AGAAGGATAAGTACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGC AGAAGGATAAGTACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCA TGGCGAGCGGGGTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGC TGGCGAGCGGGGTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGC ITGGACAGTTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATG ATGGACAGTTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATG CAGGACCAGCTGGGCTACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCG CAGGACCAGCTGGGCTACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCG CAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATO CAGATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATG ACCAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGC AGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGA AGCAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGA GGCCAGCTCCAGCCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGC GGCCAGCTCCAGCCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGC CAGGCCGGGGACCTCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTG CGGAACCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCG CCGGAACCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGC CCGGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCG GCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATO GCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATC CCGGCCCACTCGAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTT CCGGCCCACTCGAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTT CGCGTCTGGCCCGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAA CGCGTCTGGCCCGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAA TAACCGCTGGCGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCC TAACCGCTGGCGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCC GGCCGCCCCTATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGG GGCCGCCCCTATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGC GGAGCCGGTGCGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAG GGAGCCGGTGCGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAG ACCGAGGAGTTCAACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGA ACCGAGGAGTTCAACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGA CATCCTCCGGAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTO CCCATCCTCCGGAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTC TTCGTCGTCGCCGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTC TTCGTCGTCGCCGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTC ACCTATCCGATCCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGC ACCTATCCGATCCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGC

GGAGGCCTCCTCTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAAC GGAGGCCTCCTCTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACC TGGCGGACATCAACGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCTGC GGCCAGAATTGGACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTG GGCCAGAATTGGACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTG CGGGCTGATGGGCAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGO AGTACGGCAGCCCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGO AGTACGGCAGCCCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCC CCCGGTGGTGGTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAA ACCCGGTGGTGGTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGA TCAAGCAGGAGGCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAG TCAAGCAGGAGGCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAG CAATGGCCACCGGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAG CAATGGCCACCGGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAG CAGGACTACCCCGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCA CAGGACTACCCCGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCA CCCTGCCCTGCCGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCAT CCCTGCCCTGCCGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCAT CCTTCCTGCCCGATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCT ATGCCACCCGGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACG CATGCCACCCGGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACG ATCGTGGCCCCGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCG6 ATCGTGGCCCCGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGG AAAACCTACACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAG CAAAACCTACACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGG TGAGAAACCTTACCACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCA GATGAACTGACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTG GATGAACTGACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGC AAAAATGCGACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAG CAAAAATGCGACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAGE AGGCATTTT

[00517] OCT4-2A-SOX2-2A-KLF4 (non-limiting example of an amino acid sequence

(SEQ ID NO: 39):

IAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSE MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSE VWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASP VWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASP EPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGL EPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGL TLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETL TLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETL VQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQ KGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPE KGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPE AFPPVSVTTLGSPMHSNASGSGATNFSLLKQAGDVEENPGPACMYNMMETE GEAFPPVSVTTLGSPMHSNASGSGATNFSLLKQAGDVEENPGPACMYNMMETELKP PGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPK PGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQRRKMAQENPK MHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMK MHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRRKTKTLMK DKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYS KDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNGSYSMM QDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYS QDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSMSYS QQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPE QQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVPE AAPSRLHMSQHYQSGPVPGTAINGTLPLSHMACGSGEGRGSLLTCGDVEENPGPLE PAAPSRLHMSQHYQSGPVPGTAINGTLPLSHMACGSGEGRGSLLTCGDVEENPGPLE AVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDI MAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDL AAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAAT AAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAAT VSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLLYGRESAPPPT VSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLLYGRESAPPPT APFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLSAPGS APFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLSAPGS YGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGH EYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGH RPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQM RPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQM QPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSH LKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSR LKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSR SDHLALHMKRHE SDHLALHMKRHF

[00518] Human OCT4 nucleic acid sequence (non-limiting example of a nucleic acid

sequence encoding human OCT4) (SEQ ID NO: 40):

ATGGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAG ATGGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAG GTGATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAA CTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGC GCTTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCT CTGAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGAT CTGAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGAT GGCGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGG GGCGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGA GACCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGG GGCCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGA GAAGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAG GAAGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAG AACTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATA AACTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATA CACAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCA CACAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCA AACGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAG CTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCT CTGCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTE CAGGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAA CAGGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAAC CAGTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCC CAGTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCC AAACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGA GAAACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAA GGATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAG GGATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAG CAGCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGG CAGCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGG GGACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATG GGACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATG GAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAA GGAGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGC CTTTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAAC wo 2020/069373 WO PCT/US2019/053545

[00519] Human OCT4 amino acid sequence (non-limiting example of an amino acid

sequence encoding human OCT4) (SEQ ID NO: 41):

[AGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSE MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPGSE VWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGAS VWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASP EPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGL EPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGL LGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETE TLGVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETL VQARKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQ KGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFF KGKRSSSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPE GEAFPPVSVTTLGSPMHSN

[00520] Human SOX2 nucleic acid sequence (non-limiting example of a nucleic acid

sequence encoding human SOX2) (SEQ ID NO: 42):

ATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAACTTCG ATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAACTTCG GGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGCGGCAACCAGAAAAA GGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGCGGCAACCAGAAAA CAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATGGTGTGGTCCCGCG4 CAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATGGTGTGGTCCCGCGG GCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATO GCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAGATCA GCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGACGGAGAAGCGGCCGT GCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGACGGAGAAGCGGCCGT TCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGAAGGAGCACCCGGATT ATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAGTACA CGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATGGCGAGCGGGGTCG GGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCATGGACAGTTACGCG0 GGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCATGGACAGTTACGCGC ACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGACCAGCTGGGCT ACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGACCAGCTGGGCT ACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGO ACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCCATGC ACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCT ACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAGACCT ACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCT ACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCCCTG GCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAGGCCAGCTCCAGCCC GCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAGGCCAGCTCCAGCCC CCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGACCTC CGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCCGGAACCCGCCGCC CCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCAC CCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCCGGTGCCCGGCACG GCCATTAACGGCACACTGCCCCTCTCACACATO GCCATTAACGGCACACTGCCCCTCTCACACATG

[00521] Human SOX2 amino acid sequence (non-limiting example of an amino acid

sequence encoding human SOX2) (SEQ ID NO: 43):

MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSR MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRG QRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKY RPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMN WSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQT GWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYM GSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMI NGSPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMI SMYLPGAEVPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM

[00522] Human KLF4 (non-limiting example of a nucleotide sequence encoding human

KLF4) (SEQ ID NO: 44):

ATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTCGCGTCTGGCCCG ATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTCGCGTCTGGCCCGG CGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAATAACCGCTGGCG CGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAATAACCGCTGGCGGE GAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCGGCCGCCCCTATG GAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCGGCCGCCCCTATG ACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCGGAGCCGGTGCG ACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCGGAGCCGGTGCG GCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGACCGAGGAGTTC GCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGACCGAGGAGTTC AACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGACCCATCCTCCGGA GTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCTTCGTCGTCGCCG GTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCTTCGTCGTCGCCG TCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCACCTATCCGATCC TCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCACCTATCCGATCC GGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCGGAGGCCTCCTCT GGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCGGAGGCCTCCTCT ATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCTGGCGGACATCAA ATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCTGGCGGACATCAA CGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCTGCGGCCAGAATTGGA CCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGGCGGGCTGATGGG CCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGGCGGGCTGATGGG CAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGCGAGTACGGCAGCCC GTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCACCCGGTGGTGGT GTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCACCCGGTGGTGGT GGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGATCAAGCAGGAGG GGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGATCAAGCAGGAGGC GGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAGCAATGGCCACCGG CGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGCAGGACTACCCC CCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGCAGGACTACCCCG ACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCACCCTGCCCTGCCG ACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCACCCTGCCCTGCCG CTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCATCCTTCCTGCCCGA TCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTCATGCCACCCGGT TCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTCATGCCACCCGGT TCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGATCGTGGCCCCGG TCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGATCGTGGCCCCGG AAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGCAAAACCTACACA AAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGTGAGAAACCTTAC AAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGTGAGAAACCTTAC CACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCAGATGAACTGACC CACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCAGATGAACTGACC AGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGCCAAAAATGCGAC CGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAGAGGCATTT CGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAGAGGCATTTT

[00523] Human

[00523] Human KLF4(non-limiting KLF4 (non-limiting example exampleofofanan amino acid amino sequence acid encoding sequence human human encoding

KLF4) (SEQ ID NO: 45):

AVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDL MAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVLPGRPYDL AAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAA AAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPESVAAT VSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLLYGRESAPPPT FNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLSA YGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGH EYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGH RPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQI RPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQM QPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSI QPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSH CKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSR LKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSR SDHLALHMKRHF SDHLALHMKRHE

[00524] Human RCVRN (recoverin) promoter (non-limiting example of a human RCVRN

(recoverin) promoter) (SEQ ID NO: 46):

ATTTTAATCTCACTAGGGTTCTGGGAGCACCCCCCCCCACCGCTCCCGCCCTCCA CAAAGCTCCTGGGCCCCTCCTCCCTTCAAGGATTGCGAAGAGCTGGTCGCAAATO CAAAGCTCCTGGGCCCCTCCTCCCTTCAAGGATTGCGAAGAGCTGGTCGCAAATC CTCCTAAGCCACCAGCATCTCGGTCTTCAGCTCACACCAGCCTTGAGCCCAGCCT GCGGCCAGGGGACCACGCACGTCCCACCCACCCAGCGACTCCCCAGCCGCTGCC CACTCTTCCTCACTCA CACTCTTCCTCACTCA

[00525] RSVRSV

[00525] promoter(non-limiting promoter (non-limiting example exampleofof a RSV promoter) a RSV (SEQ (SEQ promoter) ID NO: ID47): NO: 47):

AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTA0 AATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAG CAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGT CAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTA AGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGA AGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATT GGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTC GATACATAAAC

[00526] CMVCMV

[00526] promoter(non-limiting promoter (non-limiting example exampleofofa CMV promoter) a CMV (SEQ(SEQ promoter) ID NO: ID 48): NO: 48):

CATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA CATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGC GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTG 221

ACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTA ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTG ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTG CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT CCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCG TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTO GTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCA AGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGG AGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGG ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCG ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGF TGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGO TGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGC TAGAGATCCGC

[00527] EFSEFS

[00527] promoter(non-limiting promoter (non-limiting example exampleofof an an EFSEFS promoter) (SEQ (SEQ promoter) ID NO: ID49): NO: 49):

AGTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC TCGAGTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCC GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCG GAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCG GGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGT CGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGT GGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACG GGTTTGCCGCCAGAACACAGGTGTCGTGACCGCGG

[00528] Human GRK1 (rhodopsin kinase) promoter (non-limiting example of a human

promoter) (SEQ ID NO: 50):

Gggccccagaagectggtggttgtttgtccttctcaggggaaaagtgaggcggccccttggaggaaggggccg

[00529] Gggccccagaagcctggtggttgttgtccttctcaggggaaagtgaggcggcccttggaggaaggggccgg

cagaatgatctaatcggattccaagcagetcaggggattgtctttttctagcaccttcttgccactcctaagegtcctccgtgaccecg gcagaatgatctaatcggattccaagcagctcaggggattgtcttctagcaccttcttgccactcctaagcgtcctccgtgaccccgg

ctgggattcgcctggtgctgtgtcagccccggtctcccaggggcttcccagtggtccccaggaaccctcgacagggcccggtctctc

tegtecagcaagggcagggacgggccacaggecaagggo

Human

[00530] Human CRX CRX (cone (cone rod rod homeobox homeobox transcription transcription factor) factor) promoter promoter (non-limiting (non-limiting

example of a human CRX promoter) (SEQ ID NO: 51):

[00531] Gcctgtagccttaatctctcctagcagggggtttgggggagggaggaggagaaagaaagggccccttatggctga

gacacaatgacccagccacaaggagggattaccgggcg

WO wo 2020/069373 PCT/US2019/053545

[00532] Human NRL promoter (neural retina leucine zipper transcription factor enhancer

upstream of the human TK terminal promoter) (non-limiting example of a human NRL

promoter) (SEQ ID NO: 52):

[00533] Aggtaggaagtggcctttaactccatagaccctatttaaacagcttcggacaggtttaaacatctccttggataattcct Aggtaggaagtggccttaactccatagaccctattaaacagcttcggacaggttaaacatctccttggataattcct

gtatecctgttcccactcctactcagggatgatagetctaagaggtgttaggggattaggctgaaaatgtaggtcacccctcageca agtatccctgttcccactcctactcagggatgatagctctaagaggtgttaggggattaggctgaaaatgtaggtcacccctcagccatc

gggaactagaatgagtgagagaggagagaggggcagagacacacacattcgcatattaaggtgacgcgtgtggcctegaacace

gagcgaccctgcagegacccgcttaa gagcgaccctgcagcgacccgcttaa

[00534] Human

[00534] Human redopsin red opsin promoter promoter (hred (hredpromoter) promoter)(SEQ ID NO: (SEQ 101): ID NO: 101):

[00535]

[00535]atccggttccaggcctcggccctaaatagtctccctgggctttcaagagaaccacatgagaaaggaggattcg Gatccggttccaggcctcggccctaaatagtctccctgggctcaagagaaccacatgagaaaggaggattcggg

tgagcagtttcaccacccaccccccagtctgcaaatectgacccgtgggtccacctgccccaaaggcggacgcaggacagtag ctctgagcagtttcaccacccaccccccagtctgcaaatcctgacccgtgggtccacctgccccaaaggcggacgcaggacagtaga

gggaacagagaacacataaacacagagagggcacagcggctcccacagtcaccgccaccttcctggcggggatgggtggggo agggaacagagaacacataaacacagagagggccacagcggctcccacagtcaccgccacctcctggcggggatgggtggggo

gtctgagtttggttcccagcaaatecctctgagccgeccttgcgggctcgcctcaggagcaggggagcaagaggtgggaggaggag gtctgagtttggtcccagcaaatccctctgagccgccctgcgggctcgcctcaggagcaggggagcaagaggtgggaggaggag

gtctaagtcccaggcccaattaagagatcaggtagtgtagggtttgggagcttttaaggtgaagaggcccgggctgateccacagge gtctaagtcccaggcccaattaagagatcaggtagtgtagggttgggagctaaggtgaagaggcccgggctgatcccacaggcc

agtataaagcgccgtgaccctcaggtgatgcgccagggccggctgccgtcggggacagggctttccatage agtataaagcgccgtgaccctcaggtgatgcgccagggccggctgccgtcggggacagggctttccatagc

[00536] Human rhodopsin promoter (rho promoter) (SEQ ID NO: 102):

[00537] Agttaatgattaacccgccatgetacttatctacgtagecatgetctaggaagateggaattegcccttaagetago Agttaatgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagctagcag

tcttccccacctagccacctggcaaactgctccttctctcaaaggcccaaacatggcctcccagactgcaacccccaggcagtcag atctccccacctagccacctggcaaactgctccttctctcaaaggcccaaacatggcctcccagactgcaacccccaggcagtcagg

ccctgtctccacaacctcacagccaccctggacggaatctgcttcttcccacatttgagtcctcctcageccctgagetcctctgggcag

(gctgtttctttccatctttgtattcccaggggcctgcaaataaatgtttaatgaacgaacaagagagtgaattccaattccatgcaacaag ggctgtttcttccatcttgtattcccaggggcctgcaaataaatgtaatgaacgaacaagagagtgaattccaattccatgcaacaag

gattgggctcctgggccctaggctatgtgtctggcaccagaaacggaagctgcaggttgcageccctgccctcatggagetectect

cagaggagtgtggggactggatgactccagaggtaacttgtgggggaacgaacaggtaaggggctgtgtgacgagatgagag tcagaggagtgtggggactggatgactccagaggtaactgtgggggaacgaacaggtaaggggctgtgtgacgagatgagagact

gggagaataaaccagaaagtctctagctgtccagaggacatagcacagaggcccatggtccctatttcaaacccaggecaccagact gggagaataaaccagaaagtctctagctgtccagaggacatagcacagaggcccatggtccctatcaaacccaggccaccagact

gctgggaccttgggacagacaagtcatgcagaagttaggggaccttctcctcccttttcctggatggatcctgagtaccttctccto gagctgggaccttgggacagacaagtcatgcagaagttaggggaccttctcctccctttcctggatggatcctgagtacctctcctcc

etgacctcaggcttcctcctagtgtcaccttggcccctcttagaagccaattaggecctcagtitctgcagcggggattaatatgattatg ctgacctcaggcttcctcctagtgtcaccttggcccctcttagaagccaattaggccctcagttctgcagcggggattaatatgattatga

acacccccaatctcccagatgctgattcagccaggagcttaggagggggaggtcactttataagggtctgggggggtcagaacccag acacccccaatctcccagatgctgatcagccaggagcttaggagggggaggtcacttataagggtctgggggggtcagaacccag

agtcatcccctgaattctgca agtcatecctgaaattetgca

[00538] Mouse cone arrestin promoter (mcar promoter) (SEQ ID NO: 103):

[00539] Ggttcttcccatttgctacatggtctttttacctttgttccttggcctggctttggcttccagggcttctgga

ccccaacccctcccatacacatacacatgtgcactcgtgcactcaacccagcacaggataatgttcattcttgacctttccacatac tcccccccaacccctcccatacacatacacatgtgcactcgtgcactcaacccagcacaggataatgttcattcttgaccttccacatac

atctggctatgttctctctcttatctacaataaatctcctccactatacttaggagcagttatgttcttcttctttcttcttttttcattcagt

aacatcatcagaateccctagctctggcctacctcctcagtaacaatcagctgatccctggccactaatctgtactcactaatctgttttcca aacatcatcagaatcccctagctctggcctacctcctcagtacaatcagctgatccctggccactaatctgtactcactaatctgtttcca actcttggcccctgagctaattatagcagtgcttcatgccacccaccccaaccctattcttgttctctgacteccactaatetacacatt aactcttggcccctgagctaattatagcagtgcttcatgccacccaccccaaccctattcttgttctctgactcccactaatctacacattca gaggattgtggatataagaggctgggaggccagcttagcaaccagagctggag gaggattgtggatataagaggctgggaggccagcttagcaaccagagctggagg

[00540] Human

[00540] Human rhodopsin kinase rhodopsin kinase promoter promoter(hrk (hrkpromoter) (SEQ(SEQ promoter) ID NO: ID 104): NO: 104):

[00541] ggccccagaagectggtggttgtttgtccttctcaggggaaaagtgaggcggccccttggaggaagggg

[00541] Gggccccagagcctggtggttgttgtccttctcaggggaaaagtgaggcggcccttggaggaaggggccgg

gcagaatgatctaatcggattccaagcagctcaggggattgtcttttctagcaccttcttgccactcctaagcgtcctccgtgaccccgg

ctgggatttagcctggtgctgtgtcagccccggtctcccaggggctcccagtggtccccaggaaccctcgacagggcccggtctctc

tcgtccagcaagggcagggacgggccacaggccaagggc tcgtecagcaagggcagggacgggccacaggccaagggc

[00542] TRE-human OSK-SV40 (SEQ ID NO: 105):

[00543]

[00543] TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA ITATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATO CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATA GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCA CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCA AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTO AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTO ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTO GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA CGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCC CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG TAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGO AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC GGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG

CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAG CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACC GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCT CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACT GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGO GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATT GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATT AAGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG AAGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACO AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACC CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACT6 CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACTC CCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAA CCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAAC GTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCC GTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCO ATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGT TATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGT ATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTAT ATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTAT CAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAG CAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGC AGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGT AGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGT CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCGCCACCAT CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCGCCACCAT GGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGT GGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGT GATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGO GATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGC TCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCT TTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCT AGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGO GAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGG TACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAG CGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAGA CTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGG CCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGG CCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGA CCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGA AGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAA AGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAA CTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACA CTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACA CAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAAA CGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCT CGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCT GCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCA GGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAACCA GGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAACCA GTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCC GTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCGA ACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGG AACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGG ATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGO ATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCA

GCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGG GCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGG ACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGG ACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGG AGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGO AGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCT TTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCGGC TTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCGGC AGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAAC AGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAAC CCGGGCCTGCATGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGC CCCGGGCCTGCATGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGC CCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGC CCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGC GGCAACCAGAAAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATG GGCAACCAGAAAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATG GTGTGGTCCCGCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCAC ACTCGGAGATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGA AACTCGGAGATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGACG GAGAAGCGGCCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGAAG GAGAAGCGGCCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGAAG |AGCACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAG GAGCACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAG AAGGATAAGTACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATG AAGGATAAGTACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATG GCGAGCGGGGTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCAT GCGAGCGGGGTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCAI GACAGTTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCA GGACCAGCTGGGCTACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCGO IGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGA GATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGAC CAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAC CAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAG CAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAV CAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAGG CCAGCTCCAGCCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCA CCAGCTCCAGCCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCA GGCCGGGGACCTCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCC GGCCGGGGACCTCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCC GAACCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCC GGAACCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCC GGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCGG GGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCGG CTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC CTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC GGGCCCACTCGAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTC CGGCCCACTCGAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTC GCGTCTGGCCCGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAAT GCGTCTGGCCCGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAAT AACCGCTGGCGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCC AACCGCTGGCGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCG GCCGCCCCTATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCG GAGCCGGTGCGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGA GAGCCGGTGCGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGA CCGAGGAGTTCAACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGAC CCATCCTCCGGAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCT CCATCCTCCGGAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCT GTCGTCGCCGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCT TCGTCGTCGCCGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCA CCTATCCGATCCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCG CCTATCCGATCCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCG GAGGCCTCCTCTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCT GAGGCCTCCTCTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCT

GGCGGACATCAACGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCTGC GGCGGACATCAACGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCTGCG CCAGAATTGGACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGG GCCAGAATTGGACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGG CGGGCTGATGGGCAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGCGA GTACGGCAGCCCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCA GTACGGCAGCCCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCA CCCGGTGGTGGTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGAT CCCGGTGGTGGTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGAT AAGCAGGAGGCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAC CAAGCAGGAGGCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAGC AATGGCCACCGGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGO AATGGCCACCGGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGC GGACTACCCCGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCAC AGGACTACCCCGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCAC CTGCCCTGCCGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCA CCTGCCCTGCCGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCATC TCCTGCCCGATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAG CTTCCTGCCCGATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTC ATGCCACCCGGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGA ATGCCACCCGGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGA TCGTGGCCCCGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGC TCGTGGCCCCGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGC AAAACCTACACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGT AAAACCTACACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGT GAGAAACCTTACCACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCA GATGAACTGACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGC GATGAACTGACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGC CAAAAATGCGACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGA CAAAAATGCGACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAG AGGCATTTTTAAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTA TTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATA, TTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA GCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATO AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTT ATCATGTCTGGATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAG ATCATGTCTGGATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAG CATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC CATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG GCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGA GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG GCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCG AGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCG GACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCO TGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCT TTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG TTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCC GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCG CCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG CCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTG PAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGAC ATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCT GTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAA TGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAA GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAAT TTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCG TTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCG GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGT ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGA AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTI TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAG TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAG ATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAG ATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGAT- CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAG CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA GCATCTTACGGATGGCATGACAGTAAGAGAA GCATCTTACGGATGGCATGACAGTAAGAGAA

[00544]

[00544] EFS-human OSK-SV40 EFS-human OSK-SV40 (SEQ (SEQIDIDNO: NO:106): 106):

[00545]

[00545]TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGC CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCA AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCA' TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGC CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCO TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA ACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAG CGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG, AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC' CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTC CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAAC GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCT TCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGAG CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACT GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAG GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATT AGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAA AAGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTA/ AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACC CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTCGAGTG GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT GCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGT TGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTA TGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAG AACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAG AACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTC AACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGC CGCCAGAACACAGGTGTCGTGACGCGGGCGGCCGCGCCACCATGGCGGGACACC CGCCAGAACACAGGTGTCGTGACGCGGGCGGCCGCGCCACCATGGCGGGACACC GGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGTGATGGGCCAG TGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGTGATGGGCCAGG GGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGCTTCCAAGGCCC GGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGCTTCCAAGGCCC TCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCTGAGGTGTGGGG GATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGGCGTACTGTGGG CCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAGACCTCTCAGCCT CCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAGACCTCTCAGCCT AGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGGCCTCCCCGGA GAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGGCCTCCCCGGA CCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGAAGCTGGAGCA GCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGAAGCTGGAGCA AAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAACTCGAGCAATT AAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAACTCGAGCAATI GCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACACAGGCCGATO TGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACACAGGCCGATGT GGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAAACGACCATO GGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAAACGACCATCTGC CGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCTGCGGCCCTTGC TGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCAGGAGATATGCA TGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCAGGAGATATGCA AAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAACCAGTATCGAGAAC PAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCGAAACCCACAC CGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCGAAACCCACACTG CAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGGATGTGGTCCGA CAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGGATGTGGTCCGA GTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCAGCGACTATGC/ GTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCAGCGACTATGCA CAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGGACCAGTGTCC CAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGGACCAGTGTCCT TTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGGAGCCCTCACT TTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGGAGCCCTCACTI CACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCTTTCCCCCTGTCT CACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCTTTCCCCCTGTCT CTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCGGCAGCGGCGCCAC CTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCGGCAGCGGCGCCAC GAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTGC TGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAAC ATGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAAC TTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGCGGCAACCAG TTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGCGGCAACCAGA AAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATGGTGTGGTCCC AAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATGGTGTGGTCCC GCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAG GCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCACAACTCGGAG ATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGACGGAGAAGCGG ATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGACGGAGAAGCGG CCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGAAGGAGCACCCG ATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAG GATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAGAAGGATAAG TACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATGGCGAGCGGC TACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATGGCGAGCGGG GTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCATGGACAGTTAC GCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGACCAGCTO GCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCAGGACCAGCTG GGCTACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCGCAGATGCAGCCC ATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGACCAGCTCGCAG ACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCO ACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAGCAGGGCACCC TGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAGGCCAGCTCCAG CTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAGGCCAGCTCCAG CCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGAG CCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCAGGCCGGGGAC TCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCCGGAACCCGC CTCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCCGGAACCCGCC GCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCCGGTGCCCGGC GCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCCGGTGCCCGGC ACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCGGCTCCGGCGAC ACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCGGCTCCGGCGAG GGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCACTC GGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCACTC GAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTCGCGTCTGGCC GAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTCGCGTCTGGCC CGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAATAACCGCTGG CGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAATAACCGCTGG CGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCGGCCGCCCCT CGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCGGCCGCCCCT ATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCGGAGCCGGTG ATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCGGAGCCGGTG CGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGACCGAGGAGT CGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGACCGAGGAGT TCAACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGACCCATCCTCCG TCAACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGACCCATCCTCCG GAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCTTCGTCGTCGC GAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCTTCGTCGTCGC CGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCACCTATCCGAT CGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCACCTATCCGAT CCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCGGAGGCCTCCT CCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCGGAGGCCTCCT CTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCTGGCGGACATO CTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCTGGCGGACATC

AACGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCTGCGGCCAGAATTO AACGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCTGCGGCCAGAATTG GACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGGCGGGCTGATO GACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGGCGGGCTGATG GCAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGCGAGTACGGCAGC GGCAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGCGAGTACGGCAGC CGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCACCCGGTGGT CCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCACCCGGTGGTG GTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGATCAAGCAGGAC GTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGATCAAGCAGGAG GCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAGCAATGGCCA GCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGCAGGACTAG GGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGCAGGACTACCC CGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCACCCTGCCCTGC CGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCACCCTGCCCTGC CGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCATCCTTCCTGCCC CGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCATCCTTCCTGCCO ATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTCATGCCACO GATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTCATGCCACCC GGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGATCGTGGCCC GGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGATCGTGGCCC CGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGCAAAACCTAC CGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGCAAAACCTAC ACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGTGAGAAACCT ACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGTGAGAAACCT TACCACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCAGATGAACTO TACCACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCAGATGAACTG ACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGCCAAAAATGC ACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGCCAAAAATGC GACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAGAGGCATTT GACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAGAGGCATTTTT AAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTT AAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTTA TAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTT TAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTT TCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTG TCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTG GATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACG GATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACG CAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGG AGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA AGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA GGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCC GGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCG CAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAA CAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAA AACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCT AACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCT GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCO GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGG GGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT TGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT CGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAA ATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAA ATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAA AAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT AAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT "TCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA/ TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAAG GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC TGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGA CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA 231

TTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTG TTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTG CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT |AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAC GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGT TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGT TTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGG TTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGG TGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGATCCTTGAGAG TGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGT TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG TTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTG GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATAC GCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATAC ACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC ACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC GGATGGCATGACAGTAAGAGAA

[00546]

[00546] TRE-Fluc-SV40 (SEQ TRE-Fluc-SV40 ID ID (SEQ NO:NO: 107): 107):

[00547]

[00547] TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATO CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGC CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCA AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC GGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCAT TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGAT7 ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCO TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA CGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG, AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA CTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC7 CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC

GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTC TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA, GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCT CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACT GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAG GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGO CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATT GGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAA, AAGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAA AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACC CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACTO CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACTC CCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAAC GTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCC GTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCC ATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAA TATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGT ATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTAT CAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGO CAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGC GAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTT AGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGT CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCATGGAAGA CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCATGGAAGA GCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGC CGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAAC CGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAAC CGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAAC AATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTC AATTGCTTTTACAGATGCACATATCGAGGTGGACATCACTTACGCTGAGTACTTC GAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAAT GAAATGTCCGTTCGGTTGGCAGAAGCTATGAAACGATATGGGCTGAATACAAAT CACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTC CACAGAATCGTCGTATGCAGTGAAAACTCTCTTCAATTCTTTATGCCGGTGTTGG GCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAAG GCGCGTTATTTATCGGAGTTGCAGTTGCGCCCGCGAACGACATTTATAATGAACG TGAATTGCTCAACAGTATGGGCATTTCGCAGCCTACCGTGGTGTTCGTTTCCAAA AAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAA AAGGGGTTGCAAAAAATTTTGAACGTGCAAAAAAAGCTCCCAATCATCCAAAAA ATTATTATCATGGATTCTAAAACGGATTACCAGGGATTTCAGTCGATGTACACGT CGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCC TCGTCACATCTCATCTACCTCCCGGTTTTAATGAATACGATTTTGTGCCAGAGTCC TCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTC TTCGATAGGGACAAGACAATTGCACTGATCATGAACTCCTCTGGATCTACTGGTC CCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCA TGCCTAAAGGTGTCGCTCTGCCTCATAGAACTGCCTGCGTGAGATTCTCGCATGC CAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTT CAGAGATCCTATTTTTGGCAATCAAATCATTCCGGATACTGCGATTTTAAGTGTT GTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGG GTTCCATTCCATCACGGTTTTGGAATGTTTACTACACTCGGATATTTGATATGTGG

ATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCT" ATTTCGAGTCGTCTTAATGTATAGATTTGAAGAAGAGCTGTTTCTGAGGAGCCTT CAGGATTACAAGATTCAAAGTGCGCTGCTGGTGCCAACCCTATTCTCCTTCTTCG CCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTT CCAAAAGCACTCTGATTGACAAATACGATTTATCTAATTTACACGAAATTGCTTC TGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCAT TGGTGGCGCTCCCCTCTCTAAGGAAGTCGGGGAAGCGGTTGCCAAGAGGTTCCAT TGCCAGGTATCAGGCAAGGATATGGGCTCACTGAGACTACATCAGCTATTCTGA TACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATT TTACACCCGAGGGGGATGATAAACCGGGCGCGGTCGGTAAAGTTGTTCCATTTTT GAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAG TGAAGCGAAGGTTGTGGATCTGGATACCGGGAAAACGCTGGGCGTTAATCAAAG AGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCG AGGCGAACTGTGTGTGAGAGGTCCTATGATTATGTCCGGTTATGTAAACAATCCG AAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATA GAAGCGACCAACGCCTTGATTGACAAGGATGGATGGCTACATTCTGGAGACATA TTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTC GCTTACTGGGACGAAGACGAACACTTCTTCATCGTTGACCGCCTGAAGTCTCTGA TTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCA TTAAGTACAAAGGCTATCAGGTGGCTCCCGCTGAATTGGAATCCATCTTGCTCCA CACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCC ACACCCCAACATCTTCGACGCAGGTGTCGCAGGTCTTCCCGACGATGACGCCGGT GAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAA GAACTTCCCGCCGCCGTTGTTGTTTTGGAGCACGGAAAGACGATGACGGAAAAA GAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGA GAGATCGTGGATTACGTCGCCAGTCAAGTAACAACCGCGAAAAAGTTGCGCGGA GGAGTTGTGTTTGTGGACGAAGTACCGAAAGGTCTTACCGGAAAACTCGACGCA AGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGT AGAAAAATCAGAGAGATCCTCATAAAGGCCAAGAAGGGCGGAAAGATCGCCGT TAAACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTTAT GTAAACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTT CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGG CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGG ATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACGT ATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACGT AGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGA AGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGA GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAG GTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAG GTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC GTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC AGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAA AGCCTTAATTAACCTAATICACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAA CCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGC ACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTG GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT GCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCT AATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGT GAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGT GGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTT GGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTC GCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA GCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAA AAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT AAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTI TCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAA TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTF GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCC GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAAT wo WO 2020/069373 PCT/US2019/053545

TTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTGC TTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTG4 GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATG AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGT AGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGT ATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT ATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGT TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGT GCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGTT GCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGATCCTTGAGAGTT TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGG TTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGG CGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA CGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACG CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACG GATGGCATGACAGTAAGAGAA GATGGCATGACAGTAAGAGAA

[00548] shRNA against mouse KDM1a (SEQ ID NO: 108):

[00549] CACAAGTCAAACCTTTAT

[00550] shRNA against human Tet1-1 (SEQ ID NO: 109):

[00551] GGACGTAATCCAGAAAGAAGA

[00552] shRNA against human Tet1-2 (SEQ ID NO: 110):

[00553] TTGTGCCTCTGGAGGTTATAA

[00554] shRNA against human Tet3-1 (SEQ ID NO: 111):

[00555] GGAAATAAAGGCTGGTGAAGG GGAAATAAAGGCTGGTGAAGG

[00556] shRNA against human Tet3-2 (SEQ ID NO: 112):

[00557] GAAAGATGAAGGTCCATATTA

[00558] shRNA against mouse Tet1-2 (SEQ ID NO: 113):

[00559] GCAGATGGCCGTGACACAAAT GCAGATGGCCGTGACACAAAT

[00560] shRNA against mouse Tet1-1 (SEQ ID NO: 114):

[00561] GCTCATGGAGACTAGGTTTGG GCTCATGGAGACTAGGTTTGG

[00562] shRNA against both mouse and human Tet2 (SEQ ID NO: 115):

[00563] GGATGTAAGTTTGCCAGAAGC wo 2020/069373 WO PCT/US2019/053545

[00564] shRNA against mouse Tet3 (SEQ ID NO: 116):

[00565] GCTCCAACGAGAAGCTATTTG

[00566] shRNA against scramble sequence (no target in genome) (SEQ ID NO: 117):

[00567] GTTCAGATGTGCGGCGAGT

[00568] Amino acid sequence encoding P2A (SEQ ID NO: 118):

[00569] GSGATNFSLLKQAGDVEENPGP GSGATNFSLLKQAGDVEENPGP

[00570] Nucleic acid sequence encoding P2A (SEQ ID NO: 119):

[00571]

[00571] GGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTT GGCAGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGT GAAGAAAACCCCGGGCCT

[00572] Nucleic acid sequence encoding T2A (SEQ ID NO: 120)

[00573]

[00573] GGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGA GGCTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGA GGAAAATCCCGGCCCA (SEQ ID NO: 120).

[00574] SEQ ID NO: 121:

[00575]

[00575] TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA TTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTA CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG TTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGG GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC GGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATAC CAAACGACGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCA AACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTG GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC GATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGC TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG TGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGO CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTO GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTC ACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATT GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA GATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAA TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC TCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGO GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA0 GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG GCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA AGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAA

TACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCG ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCG ATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGC AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA AGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGA CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTC CGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG CCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG TGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACO CTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACC GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGC CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC GCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCT CTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACT GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGG GGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGG CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC CACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATT GGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATT AGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAA AAGGCCTTAATTAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGC GAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAAC AGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACC CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACT CGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCTTTACTC CCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGATAGAGAAC GTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTCCC TATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTTTACTC TATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGT TATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAGAACGT ATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTAT ATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCCCTAT CAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAG CAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAAGC AGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTG AGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGT CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCGCCACCAT CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAGCGGCCGCGCCACCAT GGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGT GGCGGGACACCTGGCTTCGGATTTCGCCTTCTCGCCCCCTCCAGGTGGTGGAGGT ATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGC GATGGGCCAGGGGGGCCGGAGCCGGGCTGGGTTGATCCTCGGACCTGGCTAAGC TCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCT TTCCAAGGCCCTCCTGGAGGGCCAGGAATCGGGCCGGGGGTTGGGCCAGGCTCT AGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGC GAGGTGTGGGGGATTCCCCCATGCCCCCCGCCGTATGAGTTCTGTGGGGGGATGG STACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAG CGTACTGTGGGCCCCAGGTTGGAGTGGGGCTAGTGCCCCAAGGCGGCTTGGAGA CCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGC CCTCTCAGCCTGAGGGCGAAGCAGGAGTCGGGGTGGAGAGCAACTCCGATGGGG CCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGA CCTCCCCGGAGCCCTGCACCGTCACCCCTGGTGCCGTGAAGCTGGAGAAGGAGA

WO 2020/069373 wo PCT/US2019/053545

AGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAA AGCTGGAGCAAAACCCGGAGGAGTCCCAGGACATCAAAGCTCTGCAGAAAGAA TCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACA CTCGAGCAATTTGCCAAGCTCCTGAAGCAGAAGAGGATCACCCTGGGATATACA CAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAA. CAGGCCGATGTGGGGCTCACCCTGGGGGTTCTATTTGGGAAGGTATTCAGCCAAA CGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCT CGACCATCTGCCGCTTTGAGGCTCTGCAGCTTAGCTTCAAGAACATGTGTAAGCT GCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCA GCGGCCCTTGCTGCAGAAGTGGGTGGAGGAAGCTGACAACAATGAAAATCTTCA GGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAAC GGAGATATGCAAAGCAGAAACCCTCGTGCAGGCCCGAAAGAGAAAGCGAACCA GTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCC GTATCGAGAACCGAGTGAGAGGCAACCTGGAGAATTTGTTCCTGCAGTGCCCGA AACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAC AACCCACACTGCAGCAGATCAGCCACATCGCCCAGCAGCTTGGGCTCGAGAAGG ATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCA ATGTGGTCCGAGTGTGGTTCTGTAACCGGCGCCAGAAGGGCAAGCGATCAAGCA CGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGG GCGACTATGCACAACGAGAGGATTTTGAGGCTGCTGGGTCTCCTTTCTCAGGGGG ACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGG ACCAGTGTCCTTTCCTCTGGCCCCAGGGCCCCATTTTGGTACCCCAGGCTATGGG GCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGO AGCCCTCACTTCACTGCACTGTACTCCTCGGTCCCTTTCCCTGAGGGGGAAGCCT TTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCGGC TTCCCCCTGTCTCTGTCACCACTCTGGGCTCTCCCATGCATTCAAACGCTAGCGGC AGCGGCGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAAC CCCGGGCCTGCATGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGG CCCGGGCCTGCATGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGC CCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGG CCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGC GGCAACCAGAAAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATG GGCAACCAGAAAAACAGCCCGGACCGCGTCAAGCGGCCCATGAATGCCTTCATG GTGTGGTCCCGCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCAC GTGTGGTCCCGCGGGCAGCGGCGCAAGATGGCCCAGGAGAACCCCAAGATGCAC AACTCGGAGATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGA0 AACTCGGAGATCAGCAAGCGCCTGGGCGCCGAGTGGAAACTTTTGTCGGAGACG GAGAAGCGGCCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGAAC GAGAAGCGGCCGTTCATCGACGAGGCTAAGCGGCTGCGAGCGCTGCACATGAAG GAGCACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAG GAGCACCCGGATTATAAATACCGGCCCCGGCGGAAAACCAAGACGCTCATGAAG AAGGATAAGTACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATO AAGGATAAGTACACGCTGCCCGGCGGGCTGCTGGCCCCCGGCGGCAATAGCATG CGAGCGGGGTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCA' GCGAGCGGGGTCGGGGTGGGCGCCGGCCTGGGCGCGGGCGTGAACCAGCGCAT GGACAGTTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGC GGACAGTTACGCGCACATGAACGGCTGGAGCAACGGCAGCTACAGCATGATGCA GGACCAGCTGGGCTACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCGO GGACCAGCTGGGCTACCCGCAGCACCCGGGCCTCAATGCGCACGGCGCAGCGCA GATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGAC GATGCAGCCCATGCACCGCTACGACGTGAGCGCCCTGCAGTACAACTCCATGAC CAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAC CAGCTCGCAGACCTACATGAACGGCTCGCCCACCTACAGCATGTCCTACTCGCAG AGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGA CAGGGCACCCCTGGCATGGCTCTTGGCTCCATGGGTTCGGTGGTCAAGTCCGAGG CCAGCTCCAGCCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCC. CCAGCTCCAGCCCCCCTGTGGTTACCTCTTCCTCCCACTCCAGGGCGCCCTGCCA GGCCGGGGACCTCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCC GGCCGGGGACCTCCGGGACATGATCAGCATGTATCTCCCCGGCGCCGAGGTGCC GGAACCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCC GGAACCCGCCGCCCCCAGCAGACTTCACATGTCCCAGCACTACCAGAGCGGCCC GGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCGC GGTGCCCGGCACGGCCATTAACGGCACACTGCCCCTCTCACACATGGCATGCGG CTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC CTCCGGCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCC CGGCCCACTCGAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTC CGGCCCACTCGAGATGGCTGTCAGCGACGCGCTGCTCCCATCTTTCTCCACGTTC

GCGTCTGGCCCGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAAT GCGTCTGGCCCGGCGGGAAGGGAGAAGACACTGCGTCAAGCAGGTGCCCCGAAT AACCGCTGGCGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCC AACCGCTGGCGGGAGGAGCTCTCCCACATGAAGCGACTTCCCCCAGTGCTTCCCG GCCGCCCCTATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCC GCCGCCCCTATGACCTGGCGGCGGCGACCGTGGCCACAGACCTGGAGAGCGGCG GAGCCGGTGCGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGA GAGCCGGTGCGGCTTGCGGCGGTAGCAACCTGGCGCCCCTACCTCGGAGAGAGA CCGAGGAGTTCAACGATCTCCTGGACCTGGACTTTATTCTCTCCAATTCGCTGAC CATCCTCCGGAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCT0 CCATCCTCCGGAGTCAGTGGCCGCCACCGTGTCCTCGTCAGCGTCAGCCTCCTCT CGTCGTCGCCGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCA TCGTCGTCGCCGTCGAGCAGCGGCCCTGCCAGCGCGCCCTCCACCTGCAGCTTCA CTATCCGATCCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGG CCTATCCGATCCGGGCCGGGAACGACCCGGGCGTGGCGCCGGGCGGCACGGGCG GAGGCCTCCTCTATGGCAGGGAGTCCGCTCCCCCTCCGACGGCTCCCTTCAACCT CGGACATCAACGACGTGAGCCCCTCGGGCGGCTTCGTGGCCGAGCTCCT GCCAGAATTGGACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGG GCCAGAATTGGACCCGGTGTACATTCCGCCGCAGCAGCCGCAGCCGCCAGGTGG GGGCTGATGGGCAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGG CGGGCTGATGGGCAAGTTCGTGCTGAAGGCGTCGCTGAGCGCCCCTGGCAGCGA GTACGGCAGCCCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCA GTACGGCAGCCCGTCGGTCATCAGCGTCAGCAAAGGCAGCCCTGACGGCAGCCA CCCGGTGGTGGTGGCGCCCTACAACGGCGGGCCGCCGCGCACGTGCCCCAAGAT AAGCAGGAGGCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAG CAAGCAGGAGGCGGTCTCTTCGTGCACCCACTTGGGCGCTGGACCCCCTCTCAGC AATGGCCACCGGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAG AATGGCCACCGGCCGGCTGCACACGACTTCCCCCTGGGGCGGCAGCTCCCCAGC AGGACTACCCCGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCA AGGACTACCCCGACCCTGGGTCTTGAGGAAGTGCTGAGCAGCAGGGACTGTCAC CCTGCCCTGCCGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCATO CCTGCCCTGCCGCTTCCTCCCGGCTTCCATCCCCACCCGGGGCCCAATTACCCATC CTTCCTGCCCGATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTC CTTCCTGCCCGATCAGATGCAGCCGCAAGTCCCGCCGCTCCATTACCAAGAGCTC ATGCCACCCGGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGA ATGCCACCCGGTTCCTGCATGCCAGAGGAGCCCAAGCCAAAGAGGGGAAGACGA TCGTGGCCCCGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGC TCGTGGCCCCGGAAAAGGACCGCCACCCACACTTGTGATTACGCGGGCTGCGGC AAAACCTACACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGG7 AAAACCTACACAAAGAGTTCCCATCTCAAGGCACACCTGCGAACCCACACAGGT AGAAACCTTACCACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCA GAGAAACCTTACCACTGTGACTGGGACGGCTGTGGATGGAAATTCGCCCGCTCA GATGAACTGACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGC GATGAACTGACCAGGCACTACCGTAAACACACGGGGCACCGCCCGTTCCAGTGC CAAAAATGCGACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAA CAAAAATGCGACCGAGCATTTTCCAGGTCGGACCACCTCGCCTTACACATGAAG AGGCATTTTTAAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTA AGGCATTTTTAAATGACTAGTGCGCGCAGCGGCCGACCATGGCCCAACTTGTTTA TTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA TTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTT ATCATGTCTGGATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGA ATCATGTCTGGATCTCGGTACCGGATCCAAATTCCCGATAAGGATCTTCCTAGAG CATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCC GGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG AGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCG AGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCG TGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCT

TTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACA TTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG TTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCC TTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCG GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC GCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCG CCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT CCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGT AAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC CAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTC TCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGF ATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCT TGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAA TGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAA GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAA GGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAAT TTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCG TTAACGCGAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCG GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTF TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG ATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGA AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTT GCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAG TGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAG ATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAATAGTGGTAAGAT CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAG CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTC CTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTC GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAA GCATCTTACGGATGGCATGACAGTAAGAGAA GCATCTTACGGATGGCATGACAGTAAGAGAA

[00576] Thy 1.2

[00576] Thy1.2 promoter(RGC-specific) promoter (RGC-specific) (SEQ (SEQ ID IDNO: NO:122): 122):

[00577]

[00577]ATTCAGAGACCGGGAACCAAACTAGCCTTTAAAAAACATAAGTACA AATTCAGAGACCGGGAACCAAACTAGCCTTTAAAAAACATAAGTACA GGAGCCAGCAAGATGGCTCAGTGGGTAAAGGTGCCTACCAGCAAGCCTGACAGC GGAGCCAGCAAGATGGCTCAGTGGGTAAAGGTGCCTACCAGCAAGCCTGACAGO GAGTTCAGTCCCCACGAACTACGTGGTAGGAGAGGACCAACCAACTCTG CTGAGTTCAGTCCCCACGAACTACGTGGTAGGAGAGGACCAACCAACTCTGGAA ATCTGTTCTGCAAACACATGCTCACACACACACACACAAATAGTATAAACAATTT ATCTGTTCTGCAAACACATGCTCACACACACACACACAAATAGTATAAACAATTT TAAATTTCATTTAAAAATAATTTGTAAACAAAATCATTAGCACAGGTTTTAGAAA TAAATTTCATTTAAAAATAATTTGTAAACAAAATCATTAGCACAGGTTTTAGAA GAGCCTCTTGGTGACATCAAGTTGATGCTGTAGATGGGGTATCATTCCTGAGGA GAGCCTCTTGGTGACATCAAGTTGATGCTGTAGATGGGGTATCATTCCTGAGGAC CCAAAACCGGGTCTCAGCCTTTCCCCATTCTGAGAGTTCTCTCTTTTCTCAGCCAC CCAAAACCGGGTCTCAGCCTTTCCCCATTCTGAGAGTTCTCTCTTTTCTCAGCCAC AGCTGAAGAGTAGAGTGGCTCAGCACTGGGCTCTTGAGTTCCCAAGTCCTACAA TAGCTGAAGAGTAGAGTGGCTCAGCACTGGGCTCTTGAGTTCCCAAGTCCTACAA TGGTCAGCCTGACTACTAACCAGCCATGAAGAAACAAGGAGTGGATGGGC' GTCTGCTGGGATGGGAGTGGAGTTAGTAAGTGGCCATGGATGTAATGACCCCAG GTCTGCTGGGATGGGAGTGGAGTTAGTAAGTGGCCATGGATGTAATGACCCCAG CAATGCTGGCTAGAAGGCATGCCTCCTTTCCTTGTCTGGAGACGGAACGGGAGO CAATGCTGGCTAGAAGGCATGCCTCCTTTCCTTGTCTGGAGACGGAACGGGAGG ATCATCTTGTACTCACAGAAGGGAGAACATTCTAGCTGGTTGGGCCAAAATGTO GATCATCTTGTACTCACAGAAGGGAGAACATTCTAGCTGGTTGGGCCAAAATGTG CAAGTTCACCTGGAGGTGGTGGTGCATGCTTTTAACTCCAGTACTCAGGAGGCAC CAAGTTCACCTGGAGGTGGTGGTGCATGCTTTTAACTCCAGTACTCAGGAGGCAG GGCCAGGTGGATCTCTGTGAGTTCAAGACCAGCCTGCACTATGGAGAGAGTTTTG GGCCAGGTGGATCTCTGTGAGTTCAAGACCAGCCTGCACTATGGAGAGAGTTTTG

GGACAGCCAGAGTTACACAGAAAAATCCTGGTGGAAAATCTGAAAGAAAGAGA AAAGAAAGAAAGAAAGAAAGGAAGAAAGAAAGAAAGAGTGGCAGGCAGGCA GAAAGAAAGAAAGAAAGAAAGGAAGAAAGAAAGAAAGAGTGGCAGGCAGGCA GCAGGAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAA GGCAGGAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAA ATAGGTGCGACTTCAAGATCCGGAGTTACAAGCAGAATGCACTGTTTCCCTAACA ATAGGTGCGACTTCAAGATCCGGAGTTACAAGCAGAATGCACTGTTTCCCTAACA GGGCCAAGTGTTTTGAGTAACTGAAGGTGGGCATGATGCCTGGGAAGCAGAAAC GGGCCAAGTGTTTTGAGTAACTGAAGGTGGGCATGATGCCTGGGAAGCAGAAAC AGCCAGGCAGATGCACCCCTTGCCTTGCTTCCGAAGGGCTGCAGTAGCATG AAGCCAGGCAGATGCACCCCTTGCCTTGCTTCCGAAGGGCTGCAGTAGCATGGA AAACATGGAAAACAACCAATCCATTCCCTTTGCTGATATAACAGGCTCCAAAGCC AAACATGGAAAACAACCAATCCATTCCCTTTGCTGATATAACAGGCTCCAAAGCC AAAACCTGTCACTGGAGGCTCAAGAGCAGATCTCCAGCCAAGAGGCAAAGGAAT AAAACCTGTCACTGGAGGCTCAAGAGCAGATCTCCAGCCAAGAGGCAAAGGAAT GGGGAAGCTGGAGGGCCTCCCTCTGGTTATCCAGGCTTCTGAAGGTTCAAGO GGGGGAAGCTGGAGGGCCTCCCTCTGGTTATCCAGGCTTCTGAAGGTTCAAGCA AAGAAAGGGTTACAACCTTAAAAGGAGAGCGTCCCGGGGTATGGGTAGAAGACT AAGAAAGGGTTACAACCTTAAAAGGAGAGCGTCCCGGGGTATGGGTAGAAGACT GCTCCACCCCGACCCCCAGGGTCCCTAACCGTCTTTTCCCTGGGCGAGTCAGCCC GCTCCACCCCGACCCCCAGGGTCCCTAACCGTCTTTTCCCTGGGCGAGTCAGCCC AATCACAGGACTGAGAGTGCCTCTTTAGTAGCAGCAAGCCACTTCGGACACO AATCACAGGACTGAGAGTGCCTCTTTAGTAGCAGCAAGCCACTTCGGACACCCA AATGGAACACCTCCAGTCAGCCCTCGCCGACCACCCCACCCCCTCCATCCTTTTC AATGGAACACCTCCAGTCAGCCCTCGCCGACCACCCCACCCCCTCCATCCTTTTC CCTCAGCCTCCGATTGGCTGAATCTAGAGTCCCTCCCTGCTCCCCCCTCTCTCCCC CCTCAGCCTCCGATTGGCTGAATCTAGAGTCCCTCCCTGCTCCCCCCTCTCTCCCC CCCCTGGTGAAAACTGCGGGCTTCAGCGCTGGGTGCAGCAACTGGAGGCGTT ACCCCTGGTGAAAACTGCGGGCTTCAGCGCTGGGTGCAGCAACTGGAGGCGTTG GCGCACCAGGAGGAGGCTGCAGCTAGGGGAGTCCAGGTGAGAGCAGGCCGACC GCGCACCAGGAGGAGGCTGCAGCTAGGGGAGTCCAGGTGAGAGCAGGCCGACG GGAGGGACCCGCACATGCAAGGACCGCCGCAGGGCGAGGATGCAAGCCTTCCCC GGAGGGACCCGCACATGCAAGGACCGCCGCAGGGCGAGGATGCAAGCCTTCCCC AGCTACAGTTTTGGGAAAGGATACCAGGGCGCTCCTATATGGGGGCGCGGGAAC AGCTACAGTTTTGGGAAAGGATACCAGGGCGCTCCTATATGGGGGCGCGGGAAC GGGGAAAGAAGGTGCTCCCAGGTCGAGGTGGGAGAGGAAGGCAGTGCGGG TGGGGAAAGAAGGTGCTCCCAGGTCGAGGTGGGAGAGGAAGGCAGTGCGGGGT CACGGGCTTTCTCCCTGCTAACGGACGCTTTCGAAGAGTGGGTGCCGGAGGAGA CACGGGCTTTCTCCCTGCTAACGGACGCTTTCGAAGAGTGGGTGCCGGAGGAGA ACCATGAGGAAGGACATCAAGGACAGCCTTTGGTCCCCAAGCTCAAATCGCTTT ACCATGAGGAAGGACATCAAGGACAGCCTTTGGTCCCCAAGCTCAAATCGCTTT AGTGGTGCGAATAGAGGGAGGAGGTGGGTGGCAAACTGGAGGGAGTCCCCAGO AGTGGTGCGAATAGAGGGAGGAGGTGGGTGGCAAACTGGAGGGAGTCCCCAGC GGGTGACCTCGTGGCTGGCTGGGTGCGGGGCACCGCAGGTAAGAAAACCGCAAT GGGTGACCTCGTGGCTGGCTGGGTGCGGGGCACCGCAGGTAAGAAAACCGCAAT GTTGCGGGAGGGGACTGGGTGGCAGGCGCGGGGGAGGGGAAAGCTAGAAAGGA GTTGCGGGAGGGGACTGGGTGGCAGGCGCGGGGGAGGGGAAAGCTAGAAAGGA TGCGAGGGAGCGGAGGGGGGAGGGAGCGGGAGAATCTCAACTGGTAGAGGAAG TGCGAGGGAGCGGAGGGGGGAGGGAGCGGGAGAATCTCAACTGGTAGAGGAAG ATTAAAATGAGGAAATAGCATCAGGGTGGGGTTAGCCAAGCCGGGCCTCAGGGA ATTAAAATGAGGAAATAGCATCAGGGTGGGGTTAGCCAAGCCGGGCCTCAGGGA AAGGGCGCAAAGTTTGTCTGGGTGTGGGCTTAGGTGGGCTGGGTATGAGATTCC AAGGGCGCAAAGTTTGTCTGGGTGTGGGCTTAGGTGGGCTGGGTATGAGATTCG GGGCGCCGAAAACACTGCTGCGCCTCTGCCAAATCACGCTACCCCTGTATCTAGT GGGCGCCGAAAACACTGCTGCGCCTCTGCCAAATCACGCTACCCCTGTATCTAGT CTGCCAGGCTTCTCCAGCCCCAGCCCCAATTCTTTTCTCTAGTGTTCCCCCTT6 TCTGCCAGGCTTCTCCAGCCCCAGCCCCAATTCTTTTCTCTAGTGTTCCCCCTTCC TCCCCTGAATCTCAAGCCCACACTCCCTCCTCCATAACCCACTGTTATCAAATCT CTCCCCTGAATCTCAAGCCCACACTCCCTCCTCCATAACCCACTGTTATCAAATCT AGTCATTTGCCACCCAACAACCATCAGGAGGCGGAAGCAGACGGGAGGAGTTT AAGTCATTTGCCACCCAACAACCATCAGGAGGCGGAAGCAGACGGGAGGAGTTT AGATCAACTTGGGCTACATCACGAGTTCCAGGCTCACCAAGGCTTCTTAAGGAG GAGATCAACTTGGGCTACATCACGAGTTCCAGGCTCACCAAGGCTTCTTAAGGAG ACCTTGTCTCTAAAATTAATTAATTAATTAATTAATAGTCCCCTTTCTCTGCCACA ACCTTGTCTCTAAAATTAATTAATTAATTAATTAATAGTCCCCTTTCTCTGCCACA GAACCTTGGGATCTGGCTCCTGGTCGCAGCTCCCCCCACCCCAGGCTGACATTCA GAACCTTGGGATCTGGCTCCTGGTCGCAGCTCCCCCCACCCCAGGCTGACATICA

CTGCCATAGCCCATCCGGAAATCCTAGTCTATTTCCCCATGGATCTTGAACTGCA CTGCCATAGCCCATCCGGAAATCCTAGTCTATTTCCCCATGGATCTTGAACTGCA GAGAGAATGGCAGAGTGGCCCGCCCTGTGCAAAGGATGTTCCTAGCCTAGGTO GAGAGAATGGCAGAGTGGCCCGCCCTGTGCAAAGGATGTTCCTAGCCTAGGTGG AGCTCGCGAACTCGCAGACTGTGCCTCTCTTGGGCAAGGACAGGCTAGACA0 AGCTCGCGAACTCGCAGACTGTGCCTCTCTTGGGCAAGGACAGGCTAGACAGCC GCCGGTGTGTTGAGCTAGGGCACTGTGGGGAAGGCAGAGAACCTGTGCAGGGC TGCCGGTGTGTTGAGCTAGGGCACTGTGGGGAAGGCAGAGAACCTGTGCAGGGC AGCAATGAACACAGGACCAGAAAACTGCAGCCCTAGGAACACTCAAGAGCTGG AGCAATGAACACAGGACCAGAAAACTGCAGCCCTAGGAACACTCAAGAGCTGG CATTTGCAAGCATCTCTGGCCTCCGTGCTTCTCACTCATGTCCCATGTCTTATAO CCATTTGCAAGCATCTCTGGCCTCCGTGCTTCTCACTCATGTCCCATGTCTTATAC AGGCCTCTGTGGCACCTCGCTTGCCTGATCTCATCCCTAGCCGTTAAGCTTTCTGC AGGCCTCTGTGGCACCTCGCTTGCCTGATCTCATCCCTAGCCGTTAAGCTTTCTGC ATGACTTATCACTTGGGGCATAATGCTGGATACCTACCATTTTCTTAGACCCCATO ATGACTTATCACTTGGGGCATAATGCTGGATACCTACCATTTTCTTAGACCCCATC AAATCCTATTTGAGTGTACGGTTCGGAGAACCTCATTTATCCGGTAAATGTCTT AAAATCCTATTTGAGTGTACGGTTCGGAGAACCTCATTTATCCGGTAAATGTCTF TACTCTGCTCTCAGGGAGCTGAGGCAGGACATCCTGAGATACATTGGGAGAGC TTACTCTGCTCTCAGGGAGCTGAGGCAGGACATCCTGAGATACATTGGGAGAGG AGATACAGTTTCAATAAAATAATAGGTTGGGTGGAGGTACATGCCTATAATGC AGATACAGTTTCAATAAAATAATAGGTTGGGTGGAGGTACATGCCTATAATGCC ACCACTCAGGAAATGGTGGCAGCTTCGTGAGTTTGAGGCCAACCCAAGAAACAT ACCACTCAGGAAATGGTGGCAGCTTCGTGAGTTTGAGGCCAACCCAAGAAACAT AGTGAAACCCTGTCAGTAAATAAGTAAGCAAGTATTTGAGTATCTACTATATGCT AGTGAAACCCTGTCAGTAAATAAGTAAGCAAGTATTTGAGTATCTACTATATGCT AGGGCTGACCTGGACATTAGGGGTCATCTTCTGAACAAACTAGTGCTTGAGGGA AGGGCTGACCTGGACATTAGGGGTCATCTTCTGAACAAACTAGTGCTTGAGGGA GGTATTTGGGGTTTTTGTTTGTTTAATGGATCTGAATGAGTTCCAGAGACTGGCTA CACAGCGATATGACTGAGCTTAACACCCCTAAAGCATACAGTCAGACCAATT CACAGCGATATGACTGAGCTTAACACCCCTAAAGCATACAGTCAGACCAATTAG ACAATAAAAGGTATGTATAGCTTACCAAATAAAAAAATTGTATTTTCAAGAGAG ACAATAAAAGGTATGTATAGCTTACCAAATAAAAAAATTGTATTTTCAAGAGAG GTCTGTCTGTGTAGCCCTGGCTGTTCTTGAACTCACTCTGTAGACCAGGCTGG TGTCTGTCTGTGTAGCCCTGGCTGTTCTTGAACTCACTCTGTAGACCAGGCTGGCC GGAAATCCATCTGCCTGCCTCTGCCTCTCTGCCTCTCTGCCTCTCTGCCTCTCTC TGGAAATCCATCTGCCTGCCTCTGCCTCTCTGCCTCTCTGCCTCTCTGCCTCTCTCT TGCCTCTCTCTGCCTCTCTCTGCCCCTCTCTGCCCCTCTCTGCCCCTCTCTGCCGGC CCTCTGCCTTTTGCCCTCTGCCCTCTGTTCTCTGGCCTCTGCCCTCTGCCCTCTGGC CCTCTGCCTTTTGCCCTCTGCCCTCTGTTCTCTGGCCTCTGCCCTCTGCCCTCTGGC CTCTGGCCTCTGCCTCTGCCTCTTGAGTGCTGGAATCAAAGGTGTGAGCTCTGTA CTCTGGCCTCTGCCTCTGCCTCTTGAGTGCTGGAATCAAAGGTGTGAGCTCTGTA GGTCTTAAGTTCCAGAAGAAAGTAATGAAGTCACCCAGCAGGGAGGTGCTCAG GGTCTTAAGTTCCAGAAGAAAGTAATGAAGTCACCCAGCAGGGAGGTGCTCAGG ACAGCACAGACACACACCCAGGACATAGGCTCCCACTTCCTTGGCTTTCTCTGA GACAGCACAGACACACACCCAGGACATAGGCTCCCACTTCCTTGGCTTTCTCTGA GTGGCAAAGGACCTTAGGCAGTGTCACTCCCTAAGAGAAGGGGATAAAGAGAGG GTGGCAAAGGACCTTAGGCAGTGTCACTCCCTAAGAGAAGGGGATAAAGAGAGG GGCTGAGGTATTCATCATGTGCTCCGTGGATCTCAAGCCCTCAAGGTAAATGGGG ACCCACCTGTCCTACCAGCTGGCTGACCTGTAGCTTTCCCCACCACAGAATCCAA TCGGAACTCTTGGCACCTAGAGGATCTCGAGGTCCTTCCTCTGCAGAGGTCT GTCGGAACTCTTGGCACCTAGAGGATCTCGAGGTCCTTCCTCTGCAGAGGTCTTG CTTCTCCCGGTCAGCTGACTCCCTCCCCAAGTCCTTCAAATATCTCAGAACATGO CTTCTCCCGGTCAGCTGACTCCCTCCCCAAGTCCTTCAAATATCTCAGAACATGG GAGAAACGGGGACCTTGTCCCTCCTAAGGAACCCCAGTGCTGCATGCCATCAT GGAGAAACGGGGACCTTGTCCCTCCTAAGGAACCCCAGTGCTGCATGCCATCAT CCCCCCCACCCTCGCCCCCACCCCCGCCACTTCTCCCTCCATGCATACCACTAGCT CCCCCCCACCCTCGCCCCCACCCCCGCCACTTCTCCCTCCATGCATACCACTAGCT TCATTTTGTACTCTGTATTTATTCCAGGGCTGCTTCTGATTATTTAGTTTGTTO GTCATTTTGTACTCTGTATTTATTCCAGGGCTGCTTCTGATTATTTAGTTTGTTCTT TCCCTGGAGACCTGTTAGAACATAAGGGCGTATGGTGGGTAGGGGAGGCAGGAT TCCCTGGAGACCTGTTAGAACATAAGGGCGTATGGTGGGTAGGGGAGGCAGGAT ATCAGTCCCTGGGGCGAGTTCCTCCCTGCCAACCAAGCCAGATGCCTGAAAGAG ATCAGTCCCTGGGGCGAGTTCCTCCCTGCCAACCAAGCCAGATGCCTGAAAGAG

ATATGGATGAGGGAAGTTGGACTGTGCCTGTACCTGGTACAGTCATACTCTGTTO ATATGGATGAGGGAAGTTGGACTGTGCCTGTACCTGGTACAGTCATACTCTGTTG AAAGAATCATCGGGGAGGGGGGGGGGCTCAAGAGGGGAGAGCTCTGCTGAGCO AAAGAATCATCGGGGAGGGGGGGGGGCTCAAGAGGGGAGAGCTCTGCTGAGCC TTGTGGACCATCCAATGAGGATGAGGGCTTAGATTCTACCAGGTCATTCTCAG TTTGTGGACCATCCAATGAGGATGAGGGCTTAGATTCTACCAGGTCATTCTCAGC CACCACACACAAGCGCTCTGCCATCACTGAAGAAGCCCCCTAGGGCTCTTGGGC CACCACACACAAGCGCTCTGCCATCACTGAAGAAGCCCCCTAGGGCTCTTGGGC CAGGGCACACTCAGTAAAGATGCAGGTTCAGTCAGGGAATGATGGGGAAAGGG CAGGGCACACTCAGTAAAGATGCAGGTTCAGTCAGGGAATGATGGGGAAAGGG GTAGGAGGTGGGGGAGGGATCACCCCCTCCTCTAAAACACGAGCCTGCTGTC GTAGGAGGTGGGGGAGGGATCACCCCCTCCTCTAAAACACGAGCCTGCTGTCTC CAAAGGCCTCTGCCTGTAGTGAGGGTGGCAGAAGAAGACAAGGAGCCAGAACTO CAAAGGCCTCTGCCTGTAGTGAGGGTGGCAGAAGAAGACAAGGAGCCAGAACTO GACTCCAGGATCTAAGTCCGTGCAGGAAGGGGATCCTAGAACCATCTGGTTGC TGACTCCAGGATCTAAGTCCGTGCAGGAAGGGGATCCTAGAACCATCTGGTTGG ACCCAGCTTACCAAGGGAGAGCCTTTATTCTTCTTTCCCTTGCCCCTCTGTGCCAC ACCCAGCTTACCAAGGGAGAGCCTTTATTCTTCTTTCCCTTGCCCCTCTGTGCCAG CCCCTCTTGCTGTCCCTGATCCCCCAGACAGCGAGAGTCTTGCAACCTGCCTCTTC CCCCTCTTGCTGTCCCTGATCCCCCAGACAGCGAGAGTCTTGCAACCTGCCTCTTC PAAGACCTCCTAATCTCAGGGGCAGGCGGTGGAGTGAGATCCGGCGTGCACACT CAAGACCTCCTAATCTCAGGGGCAGGCGGTGGAGTGAGATCCGGCGTGCACACT CTTGGAAGATAGCTTTCCCAAGGATCCTCTCCCCCACTGGCAGCTCTGCCTG TTTTGGAAGATAGCTTTCCCAAGGATCCTCTCCCCCACTGGCAGCTCTGCCTGTCC CATCACCATGTATAATACCACCACTGCTACAGCATCTCACCGAGGAAAGAAAAG CATCACCATGTATAATACCACCACTGCTACAGCATCTCACCGAGGAAAGAAAAC TGCACAATAAAACCAAGCCTCTGGAGTGTGTCCTGGTGTCTGTCTCTTCTGTGTC< TGCACAATAAAACCAAGCCTCTGGAGTGTGTCCTGGTGTCTGTCTCTTCTGTGTCC TGGCGTCTGTCTCTTCTGTGTTCTTCCAAGGTCAGAAACAAAAACCACACACTT TGGCGTCTGTCTCTTCTGTGTTCTTCCAAGGTCAGAAACAAAAACCACACACTTC RACCTGGATGGCTCGGCTGAGCACTTCTGTGTGCAGAAGGTCCAACCAGACTCTO AACCTGGATGGCTCGGCTGAGCACTTCTGTGTGCAGAAGGTCCAACCAGACTCTG GGGTACCCCGGCCCTCCCTATTCCCTTGCCTCCTGTCTCCCGCTTTTTATAGCTCC GGGTACCCCGGCCCTCCCTATTCCCTTGCCTCCTGTCTCCCGCTTTTTATAGCTCC CTATGCTGGGCTTCTCTGGAGAGTGAAATCTTTGCCCAAATCAATGCGCATTCTC CTATGCTGGGCTTCTCTGGAGAGTGAAATCTTTGCCCAAATCAATGCGCATTCTC CTGCTGAGTCATCTGGCGACAGCAGTTGAGTTCACCCGCCAACACATGGGCC TCTGCTGAGTCATCTGGCGACAGCAGTTGAGTTCACCCGCCAACACATGGGCCCA GCTATGTAGCCGAACCCTGGCTCTGGAAGTGCCAGGGACTTTGTGCATAAGTATG GCTATGTAGCCGAACCCTGGCTCTGGAAGTGCCAGGGACTTTGTGCATAAGTATG ACCATGCCCTTTTTTCACAGTCCTAGCTCTGCAGAAGTGCAGCCTGAAGGCO TACCATGCCCTTTTTTCACAGTCCTAGCTCTGCAGAAGTGCAGCCTGAAGGCCTG TCTGCTGAGAGGACATGCCCTGGAGCCCTGAAACAGGCACAGTGGGAGGAGGAA TCTGCTGAGAGGACATGCCCTGGAGCCCTGAAACAGGCACAGTGGGAGGAGGAA CGGAGGATGACAGGCATCAGGCCCTCAGTCCAAAAGCAACCACTTGAGAATGGG CGGAGGATGACAGGCATCAGGCCCTCAGTCCAAAAGCAACCACTTGAGAATGGG CTGGAGTACGAAACATGGGGTCCCGTCCCTGGATCCCTCCTCAAAGAGTAATA CTGGAGTACGAAACATGGGGTCCCGTCCCTGGATCCCTCCTCAAAGAGTAATAA TAAAATATAAACAGGTACCCCAGGCCGTTCTGGGTTTGGGTTGTAATGGGAT GTAAAATATAAACAGGTACCCCAGGCCGTTCTGGGTTTGGGTTGTAATGGGATCC ATTTGCAGAGAACTATTGAGACAGCCCAGCCGTACTGTGACAGGCAATGTGGGG ATTTGCAGAGAACTATTGAGACAGCCCAGCCGTACTGTGACAGGCAATGTGGGG GAGGAGGTTGAATCACTTGGTATTTAGCATGAATAGAATAATTCCCTGAACATTT AGGAGGTTGAATCACTTGGTATTTAGCATGAATAGAATAATTCCCTGAACATT ACTTAAACATCCATATCTAAATTACCACCACTCGCTCCCAGTCTTCCTGCCTTTC TTCTTAAACATCCATATCTAAATTACCACCACTCGCTCCCAGTCTTCCTGCCTTTG GCCAGCCTCCTGTCTGGCCATGCCTGAAGAAGGCTGGAGAAGCCACCCACCTO CGCCAGCCTCCTGTCTGGCCATGCCTGAAGAAGGCTGGAGAAGCCACCCACCTC GGCCATGACACTGCCAGCCACTTGGCAGGTGCAGCCAAACCTGAGCTGTCCCA AGGCCATGACACTGCCAGCCACTTGGCAGGTGCAGCCAAACCTGAGCTGTCCCA GAAAGGGACATTCTCAAGACCCAGGCACCCTGATCAGCACTGACTTGGAGCTA GAAAGGGACATTCTCAAGACCCAGGCACCCTGATCAGCACTGACTTGGAGCTAC AAGTGTCATGCCAGAAAAGTCTCTAAGAAAACCTTTTCAGGGAAAAGGGGGT AAGTGTCATGCCAGAAAAGTCTCTAAGAAAACCTTTTCAGGGAAAAGGGGGTGA TCAACACCGGGCAAGTTTGGGAAGCCCCACCCTTCGAGTGATGGAAGAGCAGA CTCAACACCGGGCAAGTTTGGGAAGCCCCACCCTTCGAGTGATGGAAGAGCAGA TAGGAAGCCTCAGAAGAGAGACACCGGCACCCAGGTAACGTTCCTCATGTGGTC

WO 2020/069373 wo PCT/US2019/053545

TCTGTCACACTAGGTGCTCTTCCCTGGACATCTCCGTGACCACACTCTCAGTTCTT TCTGTCACACTAGGTGCTCTTCCCTGGACATCTCCGTGACCACACTCTCAGTTCTT AGGGAGATGCGGGTGCTCTCTGAGGCTATCTCAGAGTTGCAGATTCTGAGGCCTA AGGGAGATGCGGGTGCTCTCTGAGGCTATCTCAGAGTTGCAGATTCTGAGGCCTA GAGTGACTACAGTCAGCCTAGGAAGCCACAGAGGACTGTGGACCAGGAGGGCA GAGTGACTACAGTCAGCCTAGGAAGCCACAGAGGACTGTGGACCAGGAGGGCA GAAGAGGAGAAGGGAAGAAAAACCATCAGATAGGACTTGCAATGAAACTAACC GAAGAGGAGAAGGGAAGAAAAACCATCAGATAGGACTTGCAATGAAACTAACC CAAGACAATCATAATGCAGACAGGAATGTTAAAGGCGTTCAGCAGC CAAGACAATCATAATGCAGACAGGAATGTTAAAGGCGTTCAGCAGC ADDITIONAL EMBODIMENTS

[00578] Embodiment 1. A method comprising:

inducing in a cell, tissue, organ and/or subject:

(i) OCT4 expression; (ii) SOX2 expression; and

(iii) KLF4 expression;

in the absence of inducing c-MYC expression.

[00579] Embodiment 2. The method of embodiment 1, wherein OCT4 expression is

induced by administering:

(i) a first engineered nucleic acid encoding OCT4 or encoding a Cas9 fusion protein

(CRISPR activator) and a guide RNA sequence targeting promoter or enhancer at

endogenous locus of Oct4, optionally wherein the first nucleic acid (e.g., engineered nucleic

acid) comprises RNA and/or DNA; (ii) a chemical agent that induces OCT4 expression;

(iii) an antibody that induces OCT4 expression; or

(iv) (iv) an engineered protein encoding OCT4,

optionally wherein OCT4 comprises a sequence that is at least 70% identical to SEQ ID NO:

2 or SEQ ID NO: 41.

[00580] Embodiment 3. The method of any one of embodiments 1-2, wherein SOX2

expression comprises administering:

(v) a second engineered nucleic acid encoding SOX2 encoding a Cas9 fusion

protein (CRISPR activator) and a guide RNA sequence targeting promoter or enhancer at

endogenous locus of SOX2, wherein the second engineered nucleic acid comprises RNA

and/or DNA; (vi) a chemical agent that induces SOX2 expression;

(vii) (vii) ananantibody antibodythat thatinduces inducesSOX2 SOX2expression; expression;oror

(viii) (viii) an an engineered engineered protein protein encoding encoding SOX2, SOX2,

optionally wherein SOX2 comprises a sequence that is at least 70% identical to SEQ ID NO:

4 or SEQ ID NO: 43.

[00581] Embodiment 4. The method of any one of embodiments 1-3, wherein KLF4

expression comprises administering:

(ix) a third engineered nucleic acid encoding KLF4 encoding a Cas9 fusion protein

(CRISPR activator) and a guide RNA sequence targeting promoter or enhancer at endogenous locus of KLF4, wherein the third nucleic acid (e.g., engineered nucleic acid) comprises RNA and/or DNA;

(ix) a chemical agent that induces KLF4 expression;

(xi) an antibody that induces KLF4 expression; or

(xii) an engineered protein encoding KLF4,

optionally wherein KLF4 comprises a sequence that is at least 70% identical to SEQ ID NO:

6 or SEQ ID NO: 45.

[00582] Embodiment 5. The method of any one of embodiments 2-4, wherein said first,

second, third engineered nucleic acids, or a combination thereof are present on an expression

vector or are not present on an expression vector, optionally wherein the first, second, third

engineered nucleic acids are mRNA or plasmid DNA.

Embodiment

[00583] Embodiment 6. The 6. The method method of embodiment of embodiment 5, wherein 5, wherein two two or three or three of said of said first, first,

second and third engineered nucleic acids are present in the same expression vector.

Embodiment

[00584] Embodiment 7. The 7. The method method of any of any one one of embodiments of embodiments 1-5,1-5, wherein wherein saidsaid first, first,

second and third engineered nucleic acids are present in separate expression vectors.

[00585] Embodiment8.

[00585] Embodiment 8. The The method method of of any anyone oneofof embodiments 5-7,5-7, embodiments wherein said said wherein

expression vector(s) include an inducible promoter operably linked to the first, second, third

engineered nucleic acids, or a combination thereof, optionally wherein said method further

comprises administering an inducing agent.

[00586] Embodiment9.

[00586] Embodiment 9. The The method method of of embodiment embodiment8 wherein saidsaid 8 wherein promoter comprises promoter a comprises a

tetracycline response element (TRE).

Embodiment

[00587] Embodiment 10. 10. The The method method of embodiment of embodiment 9, wherein 9, wherein administration administration of the of the

inducing agent comprises administering a protein or a fourth engineered nucleic acid

encoding the inducing agent, optionally wherein the fourth engineered nucleic acid is

introduced simultaneously as the first, second, and third engineered nucleic acids.

[00588] Embodiment 11. The method of embodiment 10, wherein the fourth engineered

nucleic acid is present on a separate expression vector from the first, second, and third

engineered nucleic acids.

[00589] Embodiment12.

[00589] Embodiment 12. The The method method of of embodiment embodiment10,10, wherein the the wherein fourth engineered fourth engineered nucleic acid is present on the same expression vector with at least one of the first, second, and

third engineered nucleic acids.

[00590] Embodiment 13. The method of any one of embodiments 9-12, wherein the

inducing agent is capable of inducing expression of the first, second, third engineered nucleic

acids, or a combination thereof from the inducible promoter in the presence of a tetracycline

WO wo 2020/069373 PCT/US2019/053545

and the method further comprises administering tetracycline and/or removing tetracycline,

optionally wherein the tetracycline is doxycycline.

[00591] Embodiment 14. The method of embodiment 13, wherein the inducing agent is

reverse tetracycline-controlled transactivator (rtTA).

[00592] Embodiment 15. The method of embodiment 14, wherein the rtTA is M2-rtTA or

rtTA3.

[00593] Embodiment 16. The method of embodiment 15, wherein the M2-rtTA comprises

an amino acid sequence that is at least 70% identical to SEQ ID NO: 15 or the rtTA3

comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11.

[00594] Embodiment17.

[00594] Embodiment 17. The The method method of of any anyone oneofof embodiments 9-12, embodiments wherein 9-12, the wherein the

acids, or a combination thereof from the inducible promoter in the absence of a tetracycline,

optionally, wherein the tetracycline is doxycycline.

[00595] Embodiment 18. 18. Embodiment The The method of embodiment method 17, 17, of embodiment wherein the the wherein inducing agent inducing is ais a agent

temperature, a chemical, a pH, a nucleic acid, a protein, optionally wherein the protein is a

tetracycline-controlled transactivator (tTA).

[00596] Embodiment 19. The method of any one of embodiments embodiment 11 or 13-

18, wherein the first, second, and third engineered nucleic acids are present in a first

expression vector and the fourth engineered nucleic acid is present in a second expression

vector.

[00597] Embodiment Embodiment20. 20. The The method method of of any anyone oneofof embodiments 9-19, embodiments wherein 9-19, the wherein the

promoter is a TRE3G, a TRE2 promoter, or a P tight promoter, optionally, wherein the

promoter comprises a engineered nucleic acid sequence that is at least 70% identical to SEQ

ID NO: 7, optionally, wherein the promoter comprises a engineered nucleic acid sequence

that is at least 70% identical to SEQ ID NO: 23, and optionally wherein the promoter

comprises a sequence that is at least 70% identical to SEQ ID NO: 24.

[00598] Embodiment21.

[00598] Embodiment 21. The The method method of of any anyone oneofof embodiments 1-7 1-7 embodiments or 10-20, wherein or 10-20, wherein

said expression vector(s) comprise a constitutive promoter operably linked to the first,

second, third, fourth engineered nucleic acids, or any combination thereof.

[00599] Embodiment 22. The method of embodiment 21, wherein the constitutive

promoter is operably linked to the fourth engineered nucleic acid but not to the first, second,

or third engineered nucleic acids, optionally wherein the constitutive promoter is CP1, CMV,

EF1 alpha, SV40, PGK1, Ubc, human beta actin, CAG, Ac5, polyhedrin, TEF1, GDS, CaM3

5S, Ubi, H1, and U6 promoter, or a tissue-specific promoter.

WO wo 2020/069373 PCT/US2019/053545

[00600] Embodiment 23. The method of embodiment 19-22, wherein the first expression

vector comprises the sequence provided in SEQ ID NO: 16, optionally wherein the second

expression vector comprises the sequence provided in SEQ ID NO: 31 or SEQ ID NO: 32.

[00601] Embodiment Embodiment 24. 24. The The method method of any of any one one of embodiments of embodiments 2-23, 2-23, wherein wherein at least at least

one of (i)-(xii) is delivered in a viral vector or is delivered without a viral vector, wherein the

viral vector is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus,

alphavirus, vaccinia virus, and an adeno-associated virus (AAV) vector, optionally wherein

delivery without a viral vector comprises administration of a naked nucleic acid,

electroporation, use of a nanoparticle, or use of liposomes.

[00602] Embodiment Embodiment 25. 25. The The method method of any of any one one of embodiments of embodiments 19-24, 19-24, wherein wherein the the first first

expression vector is a first viral vector, and the second expression vector is a viral vector,

optionally wherein the first and second viral vectors are AAV vectors.

[00603] Embodiment 26. The method of any one of embodiments 1-25 wherein at least

one engineered nucleic acid comprises an SV40-derived sequence including a sequence that

is at least 70% identical to SEQ ID NO: 8.

[00604] Embodiment 27. The methods of any one of embodiments 1-26, wherein OCT4,

KLF4, or SOX2 is a mammalian protein.

[00605] Embodiment 28. The method of any one of embodiments 1-27, wherein the cell

or tissue is in a subject, wherein the subject has a condition, is suspected of having a

condition, or at risk for a condition, optionally wherein the condition is selected from the

group consisting of ocular disease, aging, cancer, musculoskeletal disease, age-related

disease, a disease affecting a non-human animal and neurodegenerative disease.

[00606] Embodiment29.

[00606] Embodiment 29. The The method method of of any anyone oneofof embodiments 1-28, embodiments wherein 1-28, the wherein the

method further comprises regulating: cellular reprogramming, tissue repair, tissue survival,

tissue regeneration, tissue growth, tissue function, organ regeneration, organ survival, organ

function, disease, or any combination thereof, optionally wherein regulating comprises

inducing cellular reprogramming, reversing aging, improving tissue function, improving

organ function, tissue repair, tissue survival, tissue regeneration, tissue growth, promoting

angiogenesis, treating a disease, reducing scar formation, reducing the appearance of aging,

promoting organ regeneration, promoting organ survival, altering the taste and quality of

agricultural products derived from animals, treating a disease, or any combination thereof, ex

vivo or in vitro and optionally wherein treating a disease comprises inducing expression of

OCT4, KLF4, and/or SOX2 prior to the onset of disease or wherein treating a disease a

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disease comprises inducing expression of OCT4, KLF4, and/or SOX2 after the onset of

disease.

[00607] Embodiment 30. The method of embodiment 29, wherein the cell or tissue is from

hair, heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine,

optionally wherein the tissue is damaged or the tissue may be considered healthy but

suboptimal for performance or survival in current or future conditions.

[00608] Embodiment31.

[00608] Embodiment 31. The The method method of of any anyone oneofof embodiments 1-30, embodiments wherein 1-30, the wherein the

engineered nucleic acid further comprises a self-cleaving peptide, optionally wherein the self-

cleaving peptide is a 2A peptide that is at least 70% identical to SEQ ID NO: 9.

[00609] Embodiment32.

[00609] Embodiment 32. The The method method of of any anyone oneofof embodiments 1-31, embodiments wherein 1-31, the wherein the

engineered nucleic acid further comprises inverted terminal repeats (ITRs) flanking the first

nucleic acid, the second nucleic acid, the third nucleic acid, or a combination thereof,

optionally, wherein the distance between the ITRs is 4.7 kb or less.

[00610] Embodiment 33. An expression vector comprising:

(i) a first engineered nucleic acid encoding OCT4;

(ii) a second engineered nucleic acid encoding SOX2; and

(iii) a third engineered nucleic acid encoding KLF4;

[00611] in the absence of an engineered nucleic acid capable of expressing c-MYC.

[00612] Embodiment 34. The expression vector of embodiment 33, wherein the OCT4

protein comprises a sequence that is at least 70% identical to SEQ ID NO: 2 or SEQ ID NO:

41.

[00613] Embodiment Embodiment 35. 35. The The expression expression vector vector of any of any one one of embodiments of embodiments 33-34, 33-34,

wherein the SOX2 protein comprises a sequence that is at least 70% identical to SEQ ID NO:

4 or SEQ ID NO: 43.

[00614] Embodiment 36. The expression vector of any one of embodiments 33-35,

wherein the KLF4 protein comprises a sequence that is at least 70% identical to SEQ ID NO:

6 or SEQ ID NO: 45.

[00615] Embodiment 37. The expression vector of any one of embodiments 33-36, further

comprising an inducible promoter operably linked to the first, second, third engineered

nucleic acids, or any combination thereof.

[00616] Embodiment 38. 38. Embodiment The The expression vector expression of embodiment vector 37, 37, of embodiment wherein an inducing wherein an inducing

agent is capable of inducing expression of the first, second, third engineered nucleic acids, or

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any combination thereof from the inducible promoter in the presence of a tetracycline,

optionally wherein the tetracycline is doxycycline.

[00617] Embodiment 39. The expression vector of embodiment 38, wherein the inducing

agent is reverse tetracycline-controlled transactivator (rtTA).

[00618] Embodiment 40. The expression vector of embodiment 39, wherein the rtTA is

M2-rtTA or rtTA3.

[00619] Embodiment 41. The expression vector of embodiment 40, wherein the M2-rtTA

comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 15 or the

rtTA3 comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11.

[00620] Embodiment 42. The expression vector of any one of embodiments 38-41,

wherein the inducing agent is capable of inducing expression of the first, second, third

engineered nucleic acids, or any combination thereof from the inducible promoter in the

absence of a tetracycline, optionally, wherein the tetracycline is doxycycline.

[00621] Embodiment 43. 43. Embodiment The The expression vector expression of embodiment vector 42, 42, of embodiment wherein the the wherein inducing inducing

agent is a tetracycline-controlled transactivator (tTA).

[00622] Embodiment Embodiment 44. 44. The The expression expression vector vector of any of any one one of embodiments of embodiments 37-43, 37-43,

wherein the inducible promoter comprises a tetracycline-responsive element (TRE),

optionally, wherein the promoter is a TRE3G promoter comprising a engineered nucleic acid

sequence that is at least 70% identical to SEQ ID NO: 7, optionally, wherein the promoter

comprises a engineered nucleic acid sequence that is at least 70% identical to SEQ ID NO:

23, and optionally wherein the promoter comprises a sequence that is at least 70% identical to

SEQ ID NO: 24.

[00623] Embodiment 45. The expression vector of any one of embodiments 33-36,

wherein said expression vector(s) comprise a constitutive promoter operably linked to the

first, second, third engineered nucleic acids, or a combination thereof.

[00624] Embodiment 46. The expression vector of any one of embodiments 33-44,

wherein the expression vector comprises the sequence provided in SEQ ID NO: 16.

[00625] Embodiment 47. The expression vector of any one of embodiments 33-46,

wherein the expression vector is a viral vector, wherein the viral vector is selected from the

group consisting of a lentivirus, alphavirus, vaccinia virus, a herpes virus, a retrovirus, an

adenovirus, and an adeno-associated virus (AAV) vector.

[00626] Embodiment 48. The expression vector of any one of embodiments 33-47,

wherein at least one engineered nucleic acid comprises an SV40-derived sequence including

a sequence that is at least 70% identical to SEQ ID NO: 8.

[00627] Embodiment 49. The expression vectors of any one of embodiments 33-48,

wherein OCT4, KLF4, or SOX2 is a mammalian protein.

[00628] Embodiment Embodiment 50. 50. The The expression expression vector vector of any of any one one of embodiments of embodiments 33-49, 33-49,

wherein the expression vector further comprises a self-cleaving peptide, optionally wherein

the self-cleaving peptide is 2A peptide, optionally wherein the 2A peptide comprises a

sequence that is at least 70% identical to SEQ ID NO: 9.

[00629] Embodiment Embodiment 51. 51. The The expression expression vector vector of any of any one one of embodiments of embodiments 37-44 37-44 and and 46- 46-

50, wherein the expression vector comprises one inducible promoter.

[00630] Embodiment Embodiment 52. 52. The The expression expression vector vector of any of any one one of embodiments of embodiments 45-50, 45-50,

wherein the expression vector comprises one constitutive promoter.

[00631] Embodiment53.

[00631] Embodiment 53. The The expression expression vector vectorofof anyany oneone of embodiments 33-52,33-52, of embodiments

wherein the engineered nucleic acid further comprises inverted terminal repeats (ITRs)

flanking the first nucleic acid, the second nucleic acid, the third nucleic acid, or a

combination thereof.

[00632] Embodiment 54. The expression vector of embodiment 32, wherein the distance

between the ITRs is 4.7 kb or less.

[00633] Embodiment 55. A recombinant virus comprising the expression vector of any

one of embodiments 47-54, optionally wherein the recombinant virus is a retrovirus, an

adenovirus, an AAV, alphavirus, vaccinia virus, a herpes virus, or a lentivirus.

[00634] Embodiment 56. An engineered cell produced by any one of the methods of

embodiments 1-32, 63-66, 70-75, 81, and 85-87, optionally wherein the engineered cell

comprises the expression vector of any one of embodiments 33-54.

[00635] Embodiment Embodiment 57. 57. A composition A composition comprising comprising the,the, expression expression vector vector of any of any one one of of

embodiments 33-54, the recombinant virus of embodiment 55, the engineered cell of

embodiment 56, a chemical agent that is capable of inducing OCT4, KLF4, and/or SOX2

expression, an engineered protein selected from the group consisting of OCT4, KLF4, and/or

SOX2, an antibody capable of inducing expression of OCT4, KLF4, and/or SOX2, optionally

wherein the composition comprises a pharmaceutically acceptable carrier.

[00636] Embodiment 58. The composition of embodiment 57, further comprising a

second expression vector encoding an inducing agent, a second protein encoding an inducing

agent, or a second recombinant virus encoding an inducing agent, optionally wherein the

second expression vector is an AAV vector and/or the second recombinant virus is an AAV.

[00637] Embodiment Embodiment 59. 59. The The composition composition of embodiment of embodiment 58, 58, wherein wherein the the inducing inducing agent agent

is reverse tetracycline transactivator (rtTA) or tetracycline transactivator (tTA).

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[00638] Embodiment 60. The composition of any one of embodiments 58-59, wherein the

inducing agent is encoded by a viral vector, optionally, wherein the viral vector is selected

from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated

viral vector, and a retroviral vector.

[00639] Embodiment 61. The composition of embodiment 60, wherein the viral vector

encoding the inducing agent comprises a sequence set forth in SEQ ID NO: 31 or SEQ ID

NO: 32.

[00640] Embodiment Embodiment 62. 62. A kit A kit comprising comprising the the expression expression vector vector of any of any one one of of

embodiments 33-54, recombinant virus of embodiment 55, the engineered cell of

SOX2, an antibody capable of inducing expression of OCT4, KLF4, and/or SOX2, or the

composition of any one of embodiments 56-61.

[00641] Embodiment 63. A method of producing an engineered cell comprising the

method of any one of embodiments 1-32, thereby producing the engineered cell.

[00642] Embodiment Embodiment 64. 64. The The method method of embodiment of embodiment 63, 63, wherein wherein the the engineered engineered cellcell is is

an induced pluripotent stem cell.

[00643] Embodiment 65. The method of any one of embodiments 63-64, wherein the

engineered cell is the cell of embodiment 56.

Embodiment

[00644] Embodiment 66. 66. A method A method of producing of producing an engineered an engineered cell, cell, comprising comprising the the

method of any one of embodiments 1-32 and 63-65, wherein the engineered cell is produced

ex vivo.

[00645] Embodiment67.

[00645] Embodiment 67. The The method method of of any anyone oneofof embodiments 63-66, embodiments further 63-66, further

comprising generating an engineered tissue or engineered organ.

[00646] Embodiment68.

[00646] Embodiment 68. The The method method of of any anyone oneofof embodiments 66-67, embodiments further 66-67, further

comprising administering the engineered cell, engineered tissue, and/or engineered organ to a

subject in need thereof, optionally wherein the cell, tissue, and/or organ is from eye, ear,

brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine cell.

[00647] Embodiment69.

[00647] Embodiment 69. The The method method of of any anyone oneofof embodiments 63-68, embodiments wherein 63-68, the wherein the

method further comprises treating a disease, optionally wherein the disease is selected from

the group consisting of acute injuries, neurodegenerative diseases, chronic diseases,

proliferative diseases, ocular disease, cardiovascular diseases, genetic diseases, inflammatory

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diseases, autoimmunue diseases, neurological diseases, hematological diseases, painful

conditions, psychiatric disorders, metabolic disorders, chronic diseases, cancers, aging, age-

related diseases, and diseases affecting any tissue in a subject, optionally wherein the disease

is an ocular disease.

[00648] Embodiment 70. A method comprising: (i) activating OCT4;

(ii) activating SOX2; and

(iii) activating KLF4;

in a cell, tissue, organ, and/or subject and in the absence of activating c-Myc.

[00649] Embodiment 71. The method of embodiment 71, wherein the activating in any

one of (i)-(iii) comprises administering an antibody, protein, nucleic acid, or chemical agent.

[00650] Embodiment 72. The method of any one of embodiments 72, wherein the nucleic

acid, antibody, protein, and/or chemical agent replaces OCT4, SOX2, and/or KLF4.

[00651]

[00651] Embodiment Embodiment73. 73. The The method of embodiment method of embodiment72,72, wherein wherein the the replacing replacing comprises comprises

promoting cellular reprogramming.

[00652] Embodiment74.

[00652] Embodiment 74. The The method method of of any anyone oneofof embodiments 70-73, embodiments wherein 70-73, wherein

activating of any one of (i)-(iii) comprises replacing OCT4, SOX2, and/or KLF4, selected

from the group consisting of an antibody, a protein, a nucleic acid, and a chemical agent.

[00653]

[00653] Embodiment Embodiment75. 75. The The method of embodiment method of embodiment74,74, wherein wherein the the replacing replacing of OCT4, of OCT4,

SOX2, and/or KLF4 comprises administering a nucleic acid and/or protein encoding Tet1,

NR5A-2, Sall4, E-cadherin, NKX3-1, NANOG, and/or Tet2.

[00654] Embodiment76.

[00654] Embodiment 76. The The method method of of any anyone oneofof embodiments 1-321-32 embodiments and 70-75, and 70-75,

wherein the subject is healthy.

[00655] Embodiment77.

[00655] Embodiment 77. The The method method of of any anyone oneofof embodiments 1-321-32 embodiments and 70-76, and 70-76,

wherein the subject is a pediatric subject.

[00656] Embodiment 78. The method of any one of embodiments 1-32 and 70-76,

wherein the subject is an adult subject.

[00657] Embodiment 79. The method of any one of embodiments 28-32 and 70-78,

wherein the subject has, is suspected of having, or at risk for glaucoma.

[00658] Embodiment80.

[00658] Embodiment 80. The The method method of of any anyone oneofof embodiments 28-32 embodiments and 70-79, 28-32 and 70-79,

wherein the subject has, is suspected of having, or at risk for age-related decline in visual

acuity, and/or retinal function.

[00659] Embodiment 81. A method comprising administering a nucleic acid and/or

protein encoding Tet1 or Tet2 to a cell, tissue, organ, and/or subject.

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[00660] Embodiment 82. The method of embodiment 81, wherein the subject has a

disease.

[00661] Embodiment 83. The method of embodiment 82, wherein the disease is selected

from acute injuries, neurodegenerative diseases, chronic diseases, proliferative diseases,

ocular disease, cardiovascular diseases, genetic diseases, inflammatory diseases,

autoimmunue diseases, neurological diseases, hematological diseases, painful conditions,

psychiatric disorders, metabolic disorders, chronic diseases, cancers, aging, age-related

diseases, and diseases affecting any tissue in a subject.

[00662] Embodiment 84. The method of embodiment 83, wherein the disease is an ocular

disease.

[00663] Embodiment 85. The method of any one of embodiments 1-32 and 63-84, further

comprising activating an enhancer of reprogramming in the cell, tissue, organ and/or subject.

[00664] Embodiment 86. The method of any one of embodiments 1-32 and 63-85, further

comprising inhibiting a barrier of reprogramming in the cell, tissue, organ and/or subject.

[00665] Embodiment 87. The method of embodiment 86, wherein the barrier of

reprogramming is a DNA methyltransferase (DNMT) in the cell, tissue, organ and/or subject.

[00666] Embodiment 88. A method comprising:

inducing in a subject:

(i) OCT4 expression; (ii) SOX2 expression; and

(iii) KLF4 expression;

in the absence of inducing c-MYC expression, wherein the subject has been treated with a

chemotherapy drug.

[00667] Embodiment 89. The method of embodiment 89, wherein the chemotherapy drug

is vincristine (VCS).

[00668] Embodiment 90. A method comprising inducing in a cell, tissue, organ, and/or

subject:

(i) OCT4 expression; (ii) SOX2 expression; and

(iii) (iii) KLF4 expression;

wherein OCT4, SOX2, and KLF4 is encoded by a nucleic acid and expression of OCT4,

SOX2, and/or KLF4 is induced from a single promoter.

[00669] Embodiment 91. A method comprising:

inducing in a cell, tissue, organ and/or subject:

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(i) OCT4 expression; (ii) SOX2 expression; (iii) KLF4 expression; or

(iv) any combination of (i)-(iii),

in the absence of inducing c-MYC expression.

[00670] Embodiment 92. The method of embodiment 91, wherein the combination of (i)-

(iii) comprises (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii).

[00671] Embodiment 93. An expression vector comprising:

(i) a first engineered nucleic acid encoding OCT4;

(ii) a second engineered nucleic acid encoding SOX2;

(iii) a third engineered nucleic acid encoding KLF4; or

(iv) any combination of (i)-(iii),

in the absence of an engineered nucleic acid capable of inducing c-MYC expression.

[00672] Embodiment 94. 94. Embodiment The The expression vector expression of embodiment vector 93, 93, of embodiment wherein the the wherein

combination of (i)-(iii) comprises (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii).

[00673] Embodiment 95. 95. Embodiment A recombinant virus A recombinant comprising virus the the comprising expression vector expression of any vector of any

one of embodiments 47-54 and 93-94, optionally wherein the recombinant virus is a

retrovirus, an adenovirus, an AAV, alphavirus, vaccinia virus, a herpes virus, or a lentivirus.

[00674] Embodiment 96. 96. Embodiment An engineered cellcell An engineered produced by any produced one one by any of the methods of the of of methods

embodiments 1-32, 63-66, 70-75, 81, 85-87, and 91-92, optionally wherein the engineered

cell comprises the expression vector of any one of embodiments 33-54 and 93-94.

[00675] Embodiment 97. A composition comprising the expression vector of any one of

embodiments 33-54 and 93-94, the recombinant virus of embodiment 55 or embodiment 95,

the engineered cell of embodiment 56 or 96, a chemical agent that is capable of inducing

expression of OCT4; KLF4; SOX2; or any combination thereof, an engineered protein

selected from the group consisting of OCT4; KLF4; SOX2; or any combination thereof, an

antibody capable of inducing expression of OCT4; KLF4; SOX2; or any combination thereof,

optionally wherein the composition comprises a pharmaceutically acceptable carrier.

Embodiment

[00676] Embodiment 98. 98. A kit A kit comprising comprising the the expression expression vector vector of any of any one one of of

embodiments 33-54 and 93-94, recombinant virus of embodiment 55 or 95, the engineered

cell of embodiment 56 or 96, a chemical agent that is capable of inducing expression of

OCT4; KLF4; SOX2; or any combination thereof, an engineered protein selected from the

group consisting of OCT4; KLF4; SOX2; or any combination thereof, an antibody capable of

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inducing expression of OCT4; KLF4; SOX2; or any combination thereof, or the composition

of any one of embodiments 56-61 or 97.

[00677] Embodiment 99. A method of producing an engineered cell comprising the

method of any one of embodiments 1-32 and 91-92, thereby producing the engineered cell.

[00678] Embodiment 100. A method of producing an engineered cell, comprising the

method of any one of embodiments 1-32, 63-65, 91-92, and 99, wherein the engineered cell is

produced in vivo.

[00679] Embodiment 101. A method of producing an engineered cell, comprising the

method of any one of embodiments 1-32, 63-65,91-92 and 63-65, 91-92, 99, and wherein 99, the wherein engineered the cell engineered is is cell

produced ex vivo.

[00680] Embodiment 102. A method comprising: (i) activating OCT4;

(ii) activating SOX2;

(iii) (iii) activating activatingKLF4; KLF4;oror

(iv) any combination of (i)-(iii),

in a cell, tissue, organ, subject, or any combination thereof, and in the absence of activating C- c-

Myc above endogenous levels.

[00681] Embodiment 103. The method of embodiment 102, wherein the combination of

(i)-(iii) comprises (i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii), and (iii).

[00682] Embodiment 104. A method comprising:

inducing in a subject:

(i) OCT4 expression; (ii) SOX2 expression; (iii) (iii) KLF4 expression; or

(iv) (iv) any combination of (i)-(iii),

chemotherapy drug.

[00683] Embodiment 105. The method of embodiment 104, wherein the combination of

[00684] Embodiment 106. A method comprising inducing in a cell, tissue, organ, subject,

or any combination thereof:

(i) OCT4 expression; (ii) SOX2 expression; (iii) KLF4 expression; or

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(iv) (iv) any combination of (i)-(iii),

wherein OCT4, SOX2, KLF4, or any combination thereof is encoded by a nucleic acid and

expression of OCT4, SOX2, KLF4, or any combination thereof is induced from a single

promoter.

[00685] Embodiment 107. The method of embodiment 106, wherein the combination of

Embodiment

[00686] Embodiment 108.108. The The method method of any of any one one of embodiments of embodiments 1-32, 1-32, 68-92, 68-92, or 102- or 102-

107 wherein the subject is a human.

Embodiment

[00687] Embodiment 109.109. The The method method of any of any one one of embodiments of embodiments 1-32, 1-32, 68-92, 68-92, or 102- or 102-

108, wherein the method does not induce teratoma formation.

[00688] Embodiment Embodiment 110.110. The The method method of any of any one one of embodiments of embodiments 1-32, 1-32, 68-92, 68-92, or 102- or 102-

109, wherein the method does not induce tumor formation or tumor growth.

[00689] Embodiment111.

[00689] Embodiment 111. The The method method of ofembodiment embodiment110, wherein 110, the method wherein reduces the method reduces

tumor formation or tumor growth.

[00690] Embodiment Embodiment 112.112. The The method method of any of any one one of embodiments of embodiments 1-32, 1-32, 68-92, 68-92, or 102- or 102-

111, wherein the method increases visual acuity in the subject.

[00691] Embodiment 113. The method of any one of embodiments 1-32, 68-92, or 102-

112, wherein the method does not induce cancer.

[00692] Embodiment 114. The method of any one of embodiments 1-32, 68-92, or 102-

113, wherein the method does not induce glaucoma.

Embodiment

[00693] Embodiment 115.115. The The method method of any of any one one of embodiments of embodiments 1-32, 1-32, 68-92, 68-92, or 102- or 102-

114, wherein the method reverses the epigenetic clock of the cell, the tissue, the organ, the

subject, or any combination thereof.

[00694] Embodiment116.

[00694] Embodiment 116. The The method method of ofembodiment embodiment115, wherein 115, the epigenetic wherein clock clock the epigenetic

is determined using a DNA

[00695] methylation-based (DNAm)

[00695] methylation-based (DNAm) age age estimator. estimator.

[00696] Embodiment 117. The method of any one of embodiments 1-32, 68-92, or 102-

116, wherein the method alters the expression of one or more genes associated with ageing.

[00697] Embodiment118.

[00697] Embodiment 118. The The method method of of embodiment embodiment117, wherein 117, the method wherein reduces the method reduces

expression of one or more genes associated with ageing.

[00698] Embodiment119.

[00698] Embodiment 119. The The method method of ofembodiment embodiment118, wherein 118, the method wherein reduces the method reduces

expression of 0610040J01Rik, 1700080N15Rik, 2900064F13Rik, 4833417C18Rik,

4921522P10Rik, 4930447C04Rik, 4930488N15Rik, Ace, Ackr1, Acot10, Acvr1, Adamts Adamts17,

Adralb, AI504432, Best3, Boc, Cadm3, Cand2, Ccl9, Cd14, Cd36, Cfh, Chrm3, Chrna4,

Cntn4, Cracr2b, Cryaa, CT573017.2, Cyp26a1, Cyp27a1, D330050G23Rik,

D930007P13Rik, Ddo, Dgkg, Dlk2, Dnajal-ps, Dnaja1-ps, Drd2, Dsel, Dytn, Ecscr, Edn1, Ednrb,

Efemp1, Elfn2, Epha10, Ephx1, Erbb4, Fam20a, Fbxw21, Ffar4, Flt4, Fmod, Foxp4, Fzd7,

Gabrd, Galnt15, Galnt18, Gfra2, Ggtl, Ggt1, Gm10416, Gm14964, Gm17634, Gm2065, Gm32352,

Gm33172, Gm34280, Gm35853, Gm36298, Gm36356, Gm36937, Gm3898, Gm42303,

Gm42484, Gm42537, Gm42743, Gm43151, Gm43843, Gm44545, Gm44722, Gm45516,

Gm45532, Gm47494, Gm47982, Gm47989, Gm48398, Gm48495, Gm48593, Gm48958,

Gm49089, Gm49326, Gm49331, Gm49760, Gm5796, Gm6374, Gm7276, Gm8237, Gm9796, Gm9954, Gpr75, Gprc5c, Grid2ip, Gsg112, Hapln4, Hcn3, Hcn4, Hhatl, Hs6st2,

Htr3a, Illrap, Il1rapl2, Inkal, Inka1, Kbtbd12, Kcnj11, Kcnk4, Kdelc2, Klhl33, Lame3, Lamc3, Lilra5,

Lman11, Lrfn2, Lrrc38, Lrrn4cl, Ltc4s, Manscl, Mansc1, Mir344c, Msrl, Msr1, Mycbpap, Myoc, Ngfr,

Nipal2, Olfr1372-ps1, Otop3, P2rx5, P2ry12, P4ha2, Pcdha12, Pcdha2, Pcdhac2, Pcdhb18,

Pcdhb5, Pcsk2os1, Pcsk6, Perp, Pkp1, Plxna4, Prickle2, Qsox1, Rapgef4os2, Rbp4, Rcn3,

Sec1415, Sel113, Serpinh1, Sgpp2, Shisa6, Siah3, Siglech, Slc12a4, Slc24a2, Slc2a5, Slc4a4,

Slitrk3, Smagp, Smoc2, Speer4b, Spon2, Sstr2, Sstr3, St3gal3, Stcl, Stc1, Stc2, Syndig1, Syt10,

Thsd7a, Tlr8, Tmem132a, Tmem132d, Tmem200a, Tmem44, Trpc4, Trpv4, Unc5b, Vgf,

Vmn1r90, Vwc21, Wfikkn2, Wnt11, Wnt6, Zeb2os, Zfp608, Zfp976, or any combination

thereof.

[00699] Embodiment 120. The method of embodiment 119, wherein the gene is a sensory

gene.

[00700]

[00700] Embodiment 121. The method of any one of embodiments 118-120, wherein the

gene is Ace, Kcnk4, Lamc3, Edn1, Syt10, Ngfr, Gprc5c, Cd36, Chrna4, Ednrb, Drd2, or a

combination thereof.

[00701] Embodiment 122. The method of embodiment 117, wherein the method increases

expression of one or more genes associated with ageing.

[00702] Embodiment 123. The method of any one of embodiments 1-32, 68-92, 102-122,

wherein the method increases expression of 1700031P21Rik, 1810053B23Rik,

2900045O20Rik, 2900060B14Rik, 4921504E06Rik, 4930402F11Rik, 4930453C13Rik,

4930455B14Rik, 4930500H12Rik, 4930549P19Rik, 4930555B11Rik, 4930556J02Rik,

4932442E05Rik, 4933431K23Rik, 4933438K21Rik, 6720475M21Rik, 9830132P13Rik,

A430010J10Rik, A530064D06Rik, A530065N20Rik, Abcb5, Abhd17c, AC116759.2,

AC131705.1, AC166779.3, Acot12, Adig, Akr1cl, Ankrd1, Asb15, Atp2c2, AU018091,

AW822073, Btnl10, Btn110, C130093G08Rik, C730027H18Rik, Ccdc162, Chil6, Col26a1, Corin,

Crls1, Cybrd1, Cyp2d12, Cyp7a1, D830005E20Rik, Dlx3, Dnah14, Dsc3, Dthd1, Eid2,

PCT/US2019/053545

Eps811, EU599041, Fam90a1a, Fancf, Fau-ps2, Fezf1, Gja5, Gm10248, Gm10513,

Gm10635, Gm10638, Gm10718, Gm10722, Gm10800, Gm10801, Gm11228, Gm11251,

Gm11264, Gm11337, Gm11368, Gm11485, Gm11693, Gm12793, Gm13050, Gm13066,

Gm13323, Gm13339, Gm13346, Gm13857, Gm14387, Gm14770, Gm15638, Gm16072,

Gm16161, Gm16181, Gm17200, Gm17791, Gm18025, Gm18757, Gm18795, Gm18848,

Gm19719, Gm20121, Gm20356, Gm2093, Gm21738, Gm21940, Gm22933, Gm24000,

Gm24119, Gm25394, Gm26555, Gm27047, Gm28262, Gm28530, Gm29295, Gm29825,

Gm29844, Gm3081, Gm32051, Gm32122, Gm33056, Gm33680, Gm34354, Gm34643,

Gm3551, Gm36660, Gm36948, Gm37052, Gm37142, Gm37262, Gm37535, Gm37569,

Gm37589, Gm37647, Gm37648, Gm37762, Gm38058, Gm38069, Gm38137, Gm38218,

Gm39139, Gm42535, Gm42680, Gm42895, Gm42994, Gm43027, Gm43158, Gm43288,

Gm43366, Gm44044, Gm44081, Gm44187, Gm44280, Gm44535, Gm45338, Gm45644,

Gm45740, Gm46555, Gm46565, Gm4742, Gm47485, Gm47853, Gm47992, Gm48225,

Gm48314, Gm48383, Gm48673, Gm48804, Gm48832, Gm4994, Gm5487, Gm5724,

Gm595, Gm6012, Gm6024, Gm7669, Gm7730, Gm8043, Gm8953, Gm9348, Gm9369, Gm9495, H2al2a, Ido2, Igfbp1, Kif7, Klhl31, Lrrc31, Mc5r, Mgam, Msh4, Mucl2, Mugl, Mug1,

Mybl2, Myh15, Nek10, Neurod6, Nr1h5, Olfr1042, Olfr1043, Olfr1082, Olfr1090, Olfr1124,

Olfr1167, Olfr1205, Olfr1206, Olfr1223, Olfr1263, Olfr1264, Olfr1269, Olfr127, Olfr1291-

ps1, Olfr1406, Olfr1469, Olfr215, Olfr273, Olfr328, Olfr355, Olfr372, Olfr390, Olfr427,

Olfr456, Olfr466, Olfr481, Olfr522, Olfr6, Olfr601, Olfr603, Olfr706, Olfr727, Olfr728,

Olfr741, Olfr801, Olfr812, Olfr816, Olfr822, Olfr860, Olfr890, Olfr923, Olfr943, Otogl,

Pi15, Pkhd1, Pkhd111, Platr6, Pou3f4, Prr9, Pvalb, Rhag, Sav1, Serpinb9b, Skint1, Skint3,

Skint5, Slc10a5, Slc6a4, Smok2a, Tcaf3, Tomm201, Trcg1, Trdn, Ugt1a6a, Usp171a, Usp17la,

Vmn1r178, Vmn1r179, Vmn1r33, Vmn1r74, Vmn1r87, Vmn2r102, Vmn2r113, Vmn2r17,

Vmn2r52, Vmn2r66, Vmn2r68, Vmn2r76, Vmn2r78, Wnt16, or any combination thereof.

[00703] Embodiment124.

[00703] Embodiment 124. The The method method of ofembodiment embodiment123, wherein 123, the method wherein increases the method increases

expression of Olfr816, Olfr812, Olfr1264, Olfr727, Olfr923, Olfr1090, Olfr328, Olfr1124,

Olfr522, Olfr1082, Olfr1206, Olfr1167, Olfr706, Olfr6, Pou3f4, Olfr603, Olfr127, Olfr1263,

Olfr1269, Olfr1205, Olfr390, Olfr601, Olfr860, Olfr215, Olfr741, Olfr1469, Olfr355,

Olfr481, Olfr456, Olfr1042, Olfr728, Olfr372, Olfr801, Olfr1223, Olfr822, Otogl, Olfr943,

Olfr1406, Olfr273, Olfr466, Olfr1043, Olfr427, Olfr890, Rbp4, or any combination thereof.

[00704] Embodiment 125. A method of reprogramming comprising rejuvenating the

epigenetic clock of a cell, tissue, organ, subject, or any combination thereof.

[00705] Embodiment 126. The method of embodiment 125, wherein rejuvenating the

epigenetic clock of a cell, tissue, organ, subject, or any combination thereof comprises

introducing, activating, and/or expressing OCT4, KLF4, SOX2, or any combination thereof.

[00706] Embodiment127.

[00706] Embodiment 127. The The method method of ofany anyone of of one embodiments 126,126, embodiments wherein the wherein the

epigenetic clock of a cell, tissue, organ, subject, or any combination thereof is rejuvenated to

that of a young cell, tissue, organ, subject, or any combination thereof.

[00707] Embodiment 128. The method of any one of embodiments 125-127, wherein

rejuvenating the epigenetic clock comprises altering expression of one or more genes

associated with ageing in the cell, tissue, organ, subject, or the combination thereof.

[00708] Embodiment129.

[00708] Embodiment 129. The The method method of ofembodiment embodiment128, wherein 128, the method wherein the method

comprises reducing expression of one or more genes associated with ageing.

[00709] Embodiment 130. The method of embodiment 129, wherein the method

comprises reducing expression of 0610040J01Rik, 1700080N15Rik, 2900064F13Rik,

4833417C18Rik, 4921522P10Rik, 4930447C04Rik, 4930488N15Rik, Ace, Ackr1, Acot10,

Acvrl, Acvr1, Adamts17, Adra1b, Adralb, AI504432, Best3, Boc, Cadm3, Cand2, Ccl9, Cd14, Cd36, Cfh,

Chrm3, Chrna4, Cntn4, Cracr2b, Cryaa, CT573017.2, Cyp26a1, Cyp27a1, D330050G23Rik,

Efemp1, Elfn2, Ephal0, Epha10, Ephx1, Erbb4, Fam20a, Fbxw21, Ffar4, Flt4, Fmod, Foxp4, Fzd7,

Gm33172, Gm34280, Gm35853, Gm36298, Gm36356, Gm36937, Gm3898, Gm42303,

Gm42484, Gm42537, Gm42743, Gm43151, Gm43843, Gm44545, Gm44722, Gm45516,

Gm45532, Gm47494, Gm47982, Gm47989, Gm48398, Gm48495, Gm48593, Gm48958,

Htr3a, Illrap, Illrapl2, Il1rapl2, Inkal, Kbtbd12, Kcnj11, Kcnk4, Kdelc2, Klhl33, Lamc3, Lilra5,

Lman11, Lrfn2, Lrrc38, Lrrn4cl, Ltc4s, Manscl, Mansc1, Mir344c, Msr1, Mycbpap, Myoc, Ngfr,

Thsd7a, Tlr8, Tmem132a, Tmem132d, Tmem200a, Tmem44, Trpc4, Trpv4, Unc5b, Vgf,

Vmn1r90, Vwc21, Vwc2l, Wfikkn2, Wnt11, Wnt6, Zeb2os, Zfp608, Zfp976, or any combination

thereof.

WO wo 2020/069373 PCT/US2019/053545

[00710] Embodiment 131. The method of embodiment 128-130, wherein the one or more

genes is one or more sensory genes.

[00711] Embodiment132.

[00711] Embodiment 132. The The method method of of any anyone oneofof embodiments 128-131, embodiments wherein 128-131, the wherein the

gene is Ace, Kcnk4, Lame3, Lamc3, Edn1, Syt10, Ngfr, Gprc5c, Cd36, Chrna4, Ednrb, Drd2, or a

combination thereof.

[00712] Embodiment 133. The method of any one of embodiments 128-132, wherein the

method comprises increasing expression of one or more genes associated with ageing.

[00713] Embodiment 134. The method of embodiment 133, wherein the method increases

expression of 1700031P21Rik, 1810053B23Rik, 2900045O20Rik, 2900060B14Rik,

4921504E06Rik, 4930402F11Rik, 4930453C13Rik, 4930455B14Rik, 4930500H12Rik,

4930549P19Rik, 4930555B11Rik, 4930556J02Rik, 4932442E05Rik, 4933431K23Rik,

4933438K21Rik, 6720475M21Rik, 9830132P13Rik, A430010J10Rik, A530064D06Rik,

A530065N20Rik, Abcb5, Abhd17c, AC116759.2, AC131705.1, AC166779.3, Acot12, Adig,

Akr1cl, Ankrd1, Asb15, Atp2c2, AU018091, AW822073, Btnl10, Btn110, C130093G08Rik,

C730027H18Rik, Ccdc162, Chil6, Col26a1, Corin, Crls1, Cybrd1, Cyp2d12, Cyp7a1,

D830005E20Rik, Dlx3, Dnah14, Dsc3, Dthd1, Eid2, Eps811, EU599041, Fam90a1a, Fancf,

Fau-ps2, Fezfl, Fezf1, Gja5, Gm10248, Gm10513, Gm10635, Gm10638, Gm10718, Gm10722,

Gm10800, Gm10801, Gm11228, Gm11251, Gm11264, Gm11337, Gm11368, Gm11485,

Gm11693, Gm12793, Gm13050, Gm13066, Gm13323, Gm13339, Gm13346, Gm13857,

Gm14387, Gm14770, Gm15638, Gm16072, Gm16161, Gm16181, Gm17200, Gm17791,

Gm18025, Gm18757, Gm18795, Gm18848, Gm19719, Gm20121, Gm20356, Gm2093,

Gm21738, Gm21940, Gm22933, Gm24000, Gm24119, Gm25394, Gm26555, Gm27047,

Gm28262, Gm28530, Gm29295, Gm29825, Gm29844, Gm3081, Gm32051, Gm32122,

Gm33056, Gm33680, Gm34354, Gm34643, Gm3551, Gm36660, Gm36948, Gm37052,

Gm37142, Gm37262, Gm37535, Gm37569, Gm37589, Gm37647, Gm37648, Gm37762,

Gm38058, Gm38069, Gm38137, Gm38218, Gm39139, Gm42535, Gm42680, Gm42895,

Gm42994, Gm43027, Gm43158, Gm43288, Gm43366, Gm44044, Gm44081, Gm44187,

Gm44280, Gm44535, Gm45338, Gm45644, Gm45740, Gm46555, Gm46565, Gm4742,

Gm47485, Gm47853, Gm47992, Gm48225, Gm48314, Gm48383, Gm48673, Gm48804,

Gm48832, Gm4994, Gm5487, Gm5724, Gm595, Gm6012, Gm6024, Gm7669, Gm7730, Gm8043, Gm8953, Gm9348, Gm9369, Gm9495, H2al2a, Ido2, Igfbp1, Kif7, Klhl31, Lrrc31,

Mc5r, Mgam, Msh4, Mucl2, Mug1, Mybl2, Myh15, Nek10, Neurod6, Nr1h5, Olfr1042,

Olfr1043, Olfr1082, Olfr1090, Olfr1124, Olfr1167, Olfr1205, Olfr1206, Olfr1223, Olfr1263,

Olfr1264, Olfr1269, Olfr127, Olfr1291-ps1, Olfr1406, Olfr1469, Olfr215, Olfr273, Olfr328,

WO wo 2020/069373 PCT/US2019/053545

Olfr355, Olfr372, Olfr390, Olfr427, Olfr456, Olfr466, Olfr481, Olfr522, Olfr6, Olfr601,

Olfr603, Olfr706, Olfr727, Olfr728, Olfr741, Olfr801, Olfr812, Olfr816, Olfr822, Olfr860,

Olfr890, Olfr923, Olfr943, Otogl, Pi15, Pkhd1, Pkhd111, Platr6, Pou3f4, Prr9, Pvalb, Rhag,

Sav1, Serpinb9b, Skintl, Skint1, Skint3, Skint5, Slc10a5, Slc6a4, Smok2a, Tcaf3, Tomm201, Trcgl Trcg1,

Trdn, Ugt1a6a, Ugtla6a, Usp171a, Usp17la, Vmn1r178, Vmn1r179, Vmn1r33, Vmn1r74, Vmn1r87, Vmn2r102,

Vmn2r113, Vmn2r17, Vmn2r52, Vmn2r66, Vmn2r68, Vmn2r76, Vmn2r78, Wnt16, or any

combination thereof.

[00714] Embodiment 135. The method of any one of embodiments 133-134, wherein the

method comprises increasing expression of Olfr816, Olfr812, Olfr1264, Olfr727, Olfr923,

Olfr1090, Olfr328, Olfr1124, Olfr522, Olfr1082, Olfr1206, Olfr1167, Olfr706, Olfr6,

Pou3f4, Olfr603, Olfr127, Olfr1263, Olfr1269, Olfr1205, Olfr390, Olfr601, Olfr860,

Olfr215, Olfr741, Olfr1469, Olfr355, Olfr481, Olfr456, Olfr1042, Olfr728, Olfr372, Olfr801,

Olfr1223, Olfr822, Otogl, Olfr943, Olfr1406, Olfr273, Olfr466, Olfr1043, Olfr427, Olfr890,

Rbp4, or any combination thereof.

[00715] Embodiment 136. A method of reprogramming comprising altering the

expression of one or more genes associated with ageing.

[00716] Embodiment

[00716] Embodiment137. 137. The The method of embodiment method of embodiment 136, 136, comprising comprising increasing increasing

expression of OCT4, KLF4, SOX2, or any combination thereof.

[00717]

[00717] Embodiment Embodiment138. 138. The The method of any method of anyone oneofof embodiments embodiments 136-137, 136-137, wherein wherein the the

method rejuvenates the epigenetic clock of a cell, tissue, organ, subject, or any combination

thereof.

[00718]

[00718] Embodiment 139. Embodiment TheThe 139. method of of method anyany oneone of of embodiments embodiment embodiments 136-138, embodiment 136-138,

wherein the method comprises reducing expression of one or more genes associated with

ageing.

[00719] Embodiment 140. The method of embodiment 139, wherein the method reduces

expression of 0610040J01Rik, 1700080N15Rik, 2900064F13Rik, 4833417C18Rik,

4921522P10Rik, 4930447C04Rik, 4930488N15Rik, Ace, Ackr1, Acot10, Acvrl, Acvr1, Adamts 17, Adamts17,

Cntn4, Cracr2b, Cryaa, CT573017.2, Cyp26a1, Cyp27a1, D330050G23Rik,

Gm33172, Gm34280, Gm35853, Gm36298, Gm36356, Gm36937, Gm3898, Gm42303,

Gm42484, Gm42537, Gm42743, Gm43151, Gm43843, Gm44545, Gm44722, Gm45516,

Gm45532, Gm47494, Gm47982, Gm47989, Gm48398, Gm48495, Gm48593, Gm48958,

Thsd7a, Tlr8, Tmem132a, Tmem132d, Tmem200a, Tmem44, Trpc4, Trpv4, Unc5b, Vgf,

thereof.

[00720] Embodiment 141. The method of any one of embodiments 136-140, wherein the

one or more genes is one or more sensory genes.

[00721]

[00721] Embodiment Embodiment142. 142. The The method of any method of anyone oneofof embodiments embodiments 136-140, 136-140, wherein wherein the the

combination thereof.

[00722]

[00722] Embodiment 143. Embodiment TheThe 143. method of of method anyany oneone of of embodiments embodiment embodiments 136-142, embodiment 136-142,

wherein the method comprises increasing expression of one or more genes associated with

ageing.

[00723] Embodiment Embodiment144. 144. The The method method of ofembodiment embodiment143, wherein 143, the method wherein the method

comprises increasing expression of 1700031P21Rik, 1810053B23Rik, 2900045O20Rik,

2900060B14Rik, 4921504E06Rik, 4930402F11Rik, 4930453C13Rik, 4930455B14Rik,

4930500H12Rik, 4930549P19Rik, 4930555B11Rik, 4930556J02Rik, 4932442E05Rik,

4933431K23Rik, 4933438K21Rik, 6720475M21Rik, 9830132P13Rik, A430010J10Rik,

A530064D06Rik, A530065N20Rik, Abcb5, Abhd17c, AC116759.2, AC131705.1,

AC166779.3, Acot12, Adig, Akr1cl, Ankrd1, Asb15, Atp2c2, AU018091, AW822073,

Btnl10, Btn110, C130093G08Rik, C730027H18Rik, Ccdc162, Chil6, Col26a1, Corin, Crls1, Cybrd1,

Cyp2d12, Cyp7a1, D830005E20Rik, Dlx3, Dnah14, Dsc3, Dthd1, Eid2, Eps811, EU599041,

Fam90a1a, Fancf, Fau-ps2, Fezf1, Gja5, Gm10248, Gm10513, Gm10635, Gm10638,

Gm10718, Gm10722, Gm10800, Gm10801, Gm11228, Gm11251, Gm11264, Gm11337,

Gm11368, Gm11485, Gm11693, Gm12793, Gm13050, Gm13066, Gm13323, Gm13339,

Gm13346, Gm13857, Gm14387, Gm14770, Gm15638, Gm16072, Gm16161, Gm16181,

Gm17200, Gm17791, Gm18025, Gm18757, Gm18795, Gm18848, Gm19719, Gm20121,

WO wo 2020/069373 PCT/US2019/053545

Gm20356, Gm2093, Gm21738, Gm21940, Gm22933, Gm24000, Gm24119, Gm25394,

Gm26555, Gm27047, Gm28262, Gm28530, Gm29295, Gm29825, Gm29844, Gm3081,

Gm32051, Gm32122, Gm33056, Gm33680, Gm34354, Gm34643, Gm3551, Gm36660,

Gm36948, Gm37052, Gm37142, Gm37262, Gm37535, Gm37569, Gm37589, Gm37647,

Gm37648, Gm37762, Gm38058, Gm38069, Gm38137, Gm38218, Gm39139, Gm42535,

Gm42680, Gm42895, Gm42994, Gm43027, Gm43158, Gm43288, Gm43366, Gm44044,

Gm44081, Gm44187, Gm44280, Gm44535, Gm45338, Gm45644, Gm45740, Gm46555,

Gm46565, Gm4742, Gm47485, Gm47853, Gm47992, Gm48225, Gm48314, Gm48383,

Gm48673, Gm48804, Gm48832, Gm4994, Gm5487, Gm5724, Gm595, Gm6012, Gm6024, Gm7669, Gm7730, Gm8043, Gm8953, Gm9348, Gm9369, Gm9495, H2al2a, Ido2, Igfbp1,

Kif7, Klhl31, Lrrc31, Mc5r, Mgam, Msh4, Mucl2, Mug1, Mybl2, Myh15, Nek10, Neurod6,

Nr1h5, Olfr1042, Olfr1043, Olfr1082, Olfr1090, Olfr1124, Olfr1167, Olfr1205, Olfr1206,

Olfr1223, Olfr1263, Olfr1264, Olfr1269, Olfr127, Olfr1291-ps1, Olfr1406, Olfr1469,

Olfr215, Olfr273, Olfr328, Olfr355, Olfr372, Olfr390, Olfr427, Olfr456, Olfr466, Olfr481,

Olfr522, Olfr6, Olfr601, Olfr603, Olfr706, Olfr727, Olfr728, Olfr741, Olfr801, Olfr812,

Olfr816, Olfr822, Olfr860, Olfr890, Olfr923, Olfr943, Otogl, Pi15, Pkhd1, Pkhd111, Platr6,

Pou3f4, Prr9, Pvalb, Rhag, Sav1, Serpinb9b, Skint1, Skint3, Skint5, Slc10a5, Slc6a4,

Smok2a, Tcaf3, Tomm201, Trcg1, Trdn, Ugt1a6a, Usp171a, Usp17la, Vmn1r178, Vmn1r179,

Vmn1r33, Vmn1r74, Vmn1r87, Vmn2r102, Vmn2r113, Vmn2r17, Vmn2r52, Vmn2r66,

Vmn2r68, Vmn2r76, Vmn2r78, Wnt16, or any combination thereof.

[00724] Embodiment145.

[00724] Embodiment 145. The The method method of ofembodiment embodiment144, wherein 144, the method wherein the method

comprises increasing expression of Olfr816, Olfr812, Olfr1264, Olfr727, Olfr923, Olfr1090,

Olfr328, Olfr1124, Olfr522, Olfr1082, Olfr1206, Olfr1167, Olfr706, Olfr6, Pou3f4, Olfr603,

Olfr127, Olfr1263, Olfr1269, Olfr1205, Olfr390, Olfr601, Olfr860, Olfr215, Olfr741,

Olfr1469, Olfr355, Olfr481, Olfr456, Olfr1042, Olfr728, Olfr372, Olfr801, Olfr1223,

Olfr822, Otogl, Olfr943, Olfr1406, Olfr273, Olfr466, Olfr1043, Olfr427, Olfr890, Rbp4, or

any combination thereof.

[00725] Embodiment 146. A method comprising resetting the transcriptional profile of old

cells invitro. cells in vitro.

[00726] Embodiment 147. A method comprising resetting the transcriptional profile of old

cells in vivo.

PCT/US2019/053545

[00727] Embodiment 148. A method comprising inducing in a subject:

(i) (i) OCT4 expression; (ii) SOX2 expression; and/or

(iii) KLF4 expression;

in the absence of inducing c-MYC expression, wherein the subject has, is at risk for, or is

suspected of having a condition that increases the DNA methylation-based age of a cell, of a

tissue, and/or of an organ within the subject, as compared to a control cell, a control tissue,

and/or of a control organ of a control subject that does not have the condition.

[00728] Embodiment 149. The method of embodiment 148, wherein the method reduces

the DNA methylation-based age of the cell, the tissue, the organ, and/or the subject.

[00729] Embodiment 150. A method of transdifferentiation comprising inducing in one

type of cell:

(i) (i) OCT4 expression; (ii) SOX2 expression; (iii) KLF4 expression; and

(iv) expression of a lineage determining factor,

wherein (i)-(iii) are expressed from a single vector, thereby transdifferentiating the cell into

another cell type.

[00730] Embodiment 151. A method of transdifferentiation comprising inducing in a cell:

(i) OCT4 expression; (ii) (ii) SOX2 expression; and

(iii) KLF4 expression; and

reducing expression of a lineage determining factor, wherein (i)-(iii) are expressed from a

single vector.

EQUIVALENTS AND SCOPE

[00731] In the claims articles such as "a," "an," and "the" may mean one or more than one

unless indicated to the contrary or otherwise evident from the context. Claims or descriptions

that include "or" between one or more members of a group are considered satisfied if one,

more than one, or all of the group members are present in, employed in, or otherwise relevant

to a given product or process unless indicated to the contrary or otherwise evident from the

context. The disclosure includes embodiments in which exactly one member of the group is

present in, employed in, or otherwise relevant to a given product or process. The disclosure

WO wo 2020/069373 PCT/US2019/053545

includes embodiments in which more than one, or all of the group members are present in,

employed in, or otherwise relevant to a given product or process.

[00732] Furthermore, Furthermore, the the disclosure disclosure encompasses encompasses all all variations, variations, combinations, combinations, and and

permutations in which one or more limitations, elements, clauses, and descriptive terms from

one or more of the listed claims is introduced into another claim. For example, any claim that

is dependent on another claim can be modified to include one or more limitations found in

any other claim that is dependent on the same base claim. Where elements are presented as

lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any

element(s) can be removed from the group. It should it be understood that, in general, where

the disclosure, or aspects described herein, is/are referred to as comprising particular

elements and/or features, certain embodiments described herein or aspects described herein

consist, or consist essentially of, such elements and/or features. For purposes of simplicity,

those embodiments have not been specifically set forth in haec verba herein. It is also noted

that the terms "comprising" and "containing" are intended to be open and permits the

inclusion of additional elements or steps. Where ranges are given, endpoints are included.

Furthermore, unless otherwise indicated or otherwise evident from the context and

understanding of one of ordinary skill in the art, values that are expressed as ranges can

assume any specific value or sub-range within the stated ranges in different embodiments

described herein, to the tenth of the unit of the lower limit of the range, unless the context

clearly dictates otherwise.

[00733] ThisThis applicationrefers application refers to to various various issued issuedpatents, published patents, patent published applications, patent applications,

journal articles, and other publications, all of which are incorporated herein by reference. If

there is a conflict between any of the incorporated references and the instant specification, the

specification shall control. In addition, any particular embodiment of the present disclosure

that falls within the prior art may be explicitly excluded from any one or more of the claims.

Because such embodiments are deemed to be known to one of ordinary skill in the art, they

may be excluded even if the exclusion is not set forth explicitly herein. Any particular

embodiment described herein can be excluded from any claim, for any reason, whether or not

related to the existence of prior art.

[00734] Those skilled in the art will recognize or be able to ascertain using no more than

routine experimentation many equivalents to the specific embodiments described herein. The

scope of the present embodiments described herein is not intended to be limited to the above

Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art

will appreciate that various changes and modifications to this description may be made

PCT/US2019/053545

without departing from the spirit or scope of the present disclosure, as defined in the

following claims.

Claims

CLAIMS 16 Sep 2025 What is claimed is:

1. A method of rejuvenating at least one cell, tissue, or organ in a subject in need thereof, comprising in vivo administration to the subject a composition comprising; a) a nucleic acid encoding OCT4; 2019347666

b) a nucleic acid encoding SOX2; and c) a nucleic acid encoding KLF4, wherein the composition does not comprise a nucleic acid encoding c-MYC, optionally wherein the composition does not comprise a nucleic acid encoding Nanog.

2. The method of claim 1, wherein rejuvenating at least one cell, tissue, or organ does not comprise reprogramming of at least one cell, tissue, or organ to a pluripotent state.

3. The method of claim 1 or 2, wherein the OCT4, SOX2, and/or KLF4 are expressed for a time period that is sufficient to rejuvenate at least one cell, tissue, or organ, and that is insufficient to reprogram the cell to a pluripotent state.

4. The method of any one of claims 1-3, wherein following administration of the composition to the subject, at least one rejuvenated cell, tissue, or organ does not express at least one stem cell marker.

5. The method of claim 4, wherein the stem cell marker is Esrrb, Nanog, Lin28, TRA-1- 60/TRA-1-81/TRA-2-54, SSEA1, SSEA4, or any combination thereof.

6. The compos method of any one of claims 1-5, wherein following administration of the composition to the subject, at least one rejuvenated cell, tissue, or organ expresses RBPMS, Brn3a, or any combination thereof.

7. The method of any one of claims 1-6, wherein rejuvenating at least one cell, tissue, or 16 Sep 2025

organ comprises restoring epigenetic information in at least one cell, tissue, or organ.

8. The method of any one of claims 1-7, wherein rejuvenating at least one cell, tissue, or organ comprises restoring epigenetic information lost due to aging, injury, disease, or any combination thereof in the at least one cell, tissue, or organ. 2019347666

9. The method of any one of claims 1-8, wherein rejuvenating at least one cell, tissue, or organ comprises reestablishing the epigenetic status of the cell, tissue, or organ to an epigenetic status that is similar to the status formed soon after fertilization or final differentiation.

10. The method of any one of claims 1-9, wherein the nucleic acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4 are encapsulated in at least one nanoparticle.

11. The method of any one of claims 1-10, wherein the nucleic acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4 comprise DNA or RNA.

12. The method of claim 11, wherein the DNA is plasmid DNA.

13. The method of claim 11, wherein the RNA is mRNA

14. The method of any one of claims 1-13, wherein the nucleic acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4 are present on one or more expression vectors.

15. The method of claim 14, wherein the nucleic acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4are present in the same expression vector.

16. The method of claim 14 or 15, wherein the one or more expression vector include an 16 Sep 2025

inducible promoter operably linked to any one of the nucleic acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4, or a combination thereof.

17. The method of claim 16, wherein the promoter is a TRE3G, a TRE2 promoter, or a P tight promoter. 2019347666

18. The method of claim 16 or 17, wherein said promoter comprises a tetracycline response element (TRE).

19. The method of any one of claims 14-18, wherein the expression vector comprises a self- cleaving peptide.

20. The method of claim 19, wherein the self-cleaving peptide is a 2A peptide

21. The method of any one of claims 14-20, wherein the expression vector comprises inverted terminal repeats (ITRs) flanking the nucleic acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4, or a combination thereof, and wherein the distance between the ITRs is 4.7 kb or less.

22. The method of any one of claims 1-21, wherein the composition further comprises an inducing agent or wherein the method further comprises administering to said subject an inducing agent.

23. The method of claim 22, wherein the inducing agent is a tetracycline or a reverse tetracycline-controlled transactivator (rtTA).

24. The method of claim 23, wherein the tetracycline is doxycycline.

25. The method of claim 23 or 24, wherein the rtTA is present in an expression vector.

26. The method of claim 25, wherein the expression vector does not comprise the nucleic 16 Sep 2025

acid encoding OCT4, the nucleic acid encoding SOX2, and the nucleic acid encoding KLF4.

27. The method of any one of claims 23-26, wherein the rtTA is M2-rtTA or rtTA3.

28. The method of any one of claims 14-27, wherein the expression vector is a viral vector. 2019347666

29. The method of claim 28, wherein the viral vector is a lentivirus, a retrovirus, an adenovirus, alphavirus, vaccinia virus, or an adeno-associated virus (AAV) vector.

30. The method of claim 29, wherein the AAV vector is AAV2 or AAV9.

31. The method of any one of claims 1-30, wherein the subject has, is suspected of having, or at risk for an ocular disease, aging, a cancer, musculoskeletal disease, an age-related disease, or a neurodegenerative disease.

32. The method of any one of claims 1-31, wherein the subject is a non-human mammal.

33. The method of any one of claims 1-32, wherein the subject is a human.

34. The method of any one of claims 1-33, wherein the cell, tissue, or organ is from the eye, the ear, the nose, the mouth including gum and roots of teeth, bone, the lung, the breast, udder, pancreas, stomach, esophagus, muscle including cardiac muscle, liver, blood vessel, skin including hair, heart, brain, nerve tissue, kidney, testis, prostate, penis, cloaca, fin, ovary, or intestine.

35. The method of any one of claims 1-34, wherein said administration is direct administration to the tissue or organ.

36. The method of any one of claims 1-35, wherein: i) OCT4 comprises an amino acid sequence having at least 90% identity to SEQ ID 16 Sep 2025

NO: 41; ii) SOX2 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 43; and/or iii) KLF4 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 45. 2019347666

37. The method of any one of claims 1-35, wherein: i) OCT4 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 41; ii) SOX2 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 43; and/or iii) KLF4 comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 45.

38. The method of any one of claims 1-35, wherein: i) OCT4 comprises the amino acid sequence of SEQ ID NO: 41; ii) SOX2 comprises the amino acid sequence of SEQ ID NO: 43; and/or iii) KLF4 comprises the amino acid sequence of SEQ ID NO: 45.